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CLINICAL AND 
LABORATORY TECHNIC 



McNEIL 



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CLINICAL 

AND 

LABORATORY TECMIC 



BY 



II. L. McNEIL, A.B., M.D., 

Adjunct Professor of Medicine and Instructor in Physical Diagnosis, University of 
Texas Medical School, Galveston, Texas. 



ILLUSTRATED 



ST. LOUIS 

C. V. MOSBY COMPANY 

1916 



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Copyright, 1916, By The C. V. Mosby Company 



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N3V -3 1916 
©CU446209 



Press of 

The C. V. Mosby Company 

St. Louis 



PREFACE 

The occasion for the writing of this small book has been the 
writer's experience as a teacher of medicine and of physical 
diagnosis at the bedside, and, at the same time, of clinical 
pathology in the laboratory. It is now generally accepted, 
almost as a truism, that, in order to become a competent in- 
ternist, one must first acquire a very intimate and personal 
knowledge of the medical laboratory. It is also a generally 
recognized fact that a successful medical diagnosis is best and 
most accurately reached by the systematic and scientific ap- 
plication of three different forms of examination, each of 
these forms being of practically equal importance from a 
clinical point of view. It is the application, therefore, of 
these three diagnostic procedures, with an ability to properly 
weigh and interpret any abnormal findings, which differ- 
entiates the good, mediocre and incompetent practitioner of 
medicine. This triad of history taking, physical diagnosis, 
and laboratory analysis, we have aimed to present in this 
book in as simple and practical a manner as possible. 

All of the laboratory tests mentioned here, as well as the 
routine methods described for history taking and physical 
diagnosis are in common use in the medical clinics of the John 
Sealy Hospital. With a few exceptions all of the laboratory 
tests mentioned are done as a routine measure. These excep- 
tions are, of course, analyses of the gastric and duodenal con- 
tents, the chemical examinations of the stools, blood cultures, 
Widal's, etc. 

Although this work was originally designed for the use of 
advanced students in the Medical Department of the Uni- 
versity of Texas Medical School, especially for clinical clerks, 
internes, etc., we feel that it may be of some value to the 
general practitioner or internist who does some of the com- 
mon laboratory tests himself, or who desires to familiarize 
himself with the common tests which are being used. 

5 



6 Preface 

With the exception of the technic which is described for 
the examination of the fresh gastric contents and duodenal 
contents and of the technic for the detection of the pancreatic 
ferments in the duodenal contents and stools, in suspected 
disease of the pancreas, we offer nothing in this work which 
is not to be found in other books on the same subject; the 
only difference between this book and many others being that 
all theory is omitted from its pages and only strictly practical 
details are given. Moreover the tests are presented in the 
systematic way in which they are carried out in actual prac- 
tice. 

It is presupposed, in offering this book, therefore, that the 
user is fairly well informed theoretically, both as to the mean- 
ing of the various physical signs and as to the diagnostic sig- 
nificance of the various laboratory tests which are mentioned. 
The book is not intended, in other words, for a beginner, but 
for one who has already had a good education in medicine. 

H. L. McNeil. 

Galveston, Texas. 



CONTENTS 

PART I. 

History Taking. 

PAGE 

General Considerations 11 

The History 12 



PART II. 

Physical Examination. 

Physical Examination 16 

PART III. 

Common Laboratory Methods Used in Diagnosis. 

Examination of the Urine 22 

Examination of the Blood 38 

Examination of the Sputum 47 

Examination of the Gastric Contents 52 

Examination of the Duodenal Contents 57 

Examination of. the Feces 60 

Examination of the Spinal Fluid 65 

Examination of the Serous Exudates and Transudates 68 

The Wassermann Reaction 70 

Gonococcus Fixation Test 81 

Appendix. 

Common Stains Used in the Laboratory 82 



INTRODUCTION 

Internal medicine as a science and as a practical art has its 
foundation in diagnosis. Diagnosis in turn depends upon nu- 
merous factors. It requires not only an accurate knowledge 
of anatomy or the structure of organs, but also a thorough un- 
derstanding of physiology or function in health. Departures 
from the normal in either of these constitute defect or dis- 
ease, and may have their origin in physical, chemical, bacterial 
and other causes. The recognition of these variations and their 
definite interpretation constitutes the most important steps in 
the cure of disease or in the amelioration of its effects. The 
physician who approaches this difficult and responsible under- 
taking must therefore avail himself of every avenue of in- 
formation. The experience of all modern clinicians demon- 
strates that a systematic anamnesis, a correct observation and 
interpretation of the clinical manifestations and an accurate 
knowledge of the physical signs, when properly combined with 
information obtained from approved laboratory analyses, of- 
fer the best hope of success in the diagnosis and treatment of 
disease. 

In this book Dr. McNeil happily combines the experience of 
the successful teacher at the bedside with the revelations of 
the scientific worker in the laboratory, and presents in clear 
and truthful outline the best practical methods of examination 
and clinical diagnosis. It is commended to physicians and 
students who may need or desire its stimulating suggestions. 

Marvin L. Graves. 

Medical Department, University of Texas. 



PART I. 

HISTORY TAKING. 

General Considerations. — Let the patient describe his com- 
plaints and illness as fully as he desires in his own words. 
Avoid guiding him with questions until this preliminary state- 
ment has been obtained. Having obtained a general view, 
as it were, of his illness, in this way, you are now ready to 
take the history in detail. 

Beware of medical terms and expressions as used by pa- 
tients. If a patient states, for instance, that he has had 
typhoid fever, ascertain how long he was in bed with fever 
and, unless there is a reasonable probability that it was 
typhoid, do not call it by that name in the history, but simply 
state the fact that the patient had fever of so many days' du- 
ration at such and such a time. Similarly, a patient will often 
state that he has had rheumatism or rheumatic fever, which 
on inquiry will prove to have been simply indefinite muscle 
pains or soreness. In order therefore to be certain as to the 
previous existence of rheumatic fever inquire as to whether 
the patient was confined to bed, whether the joints were swol- 
len and tender, whether there was fever, etc. Malaria, in- 
fluenza, "brain fever," and meningitis are, similarly, terms 
much abused by the laity. 

Beware of making snap diagnoses from classical symptoms 
of disease as given by patients. Neurasthenics ,or hypochon- 
driacs, particularly if they have consulted several physicians 
previously, are apt to exhibit the most perfect symptom-com- 
plexes of various interesting diseases, which complexes, un- 
fortunately, usually point to some other disease than that from 
which the patient is suffering. 

In the case of individuals of an extremely low grade of intel- 
ligence, in the case of delirious or unconscious individuals and 

11 



12 Clinical and Laboratory Teclinic 

in dealing with small children, if satisfactory histories cannot 
be obtained from the patient, do not attempt a detailed his- 
tory, but simply state what information you can get at the 
time and obtain the remainder from friends or relatives of 
the patient, if possible. 



THE HISTORY. 

I. Note name of patient, his age, whether married or single, 
his residence and place of birth, his occupation and the date 
of examination. 

II. State complaint briefly, in patient's own words. 

III. Family History. — 

1. Father and Mother. — Ask specifically in regard to fa- 
ther and mother of the patient. If either is dead or ill state 
cause of such death or illness, with age at death. If they 
are living and well, however, do not mention them in the his- 
tory. 

2. Brothers and Sisters. — Ask specifically in regard to 
brothers and sisters of the patients, whether they are dead or 
ill at the time. If dead or ill so state with cause of death 
or illness, with age at death. If living and well do not men- 
tion in the history. 

3. Marital Relations. — If patient is married inquire as 
to wife or husband, whether dead or ill, and if so, state cause 
of death or illness. Ask also as to death or illness of children 
of the patient and if any are dead or ill state cause, with age 
at death. If patient be a married woman inquire as to mis- 
carriages or abortions. 

Do not mention wife, husband or children in the history 
unless they are dead or ill. 

4. Tuberculosis. — Ask specifically of the patient regard- 
ing the presence or absence of tuberculosis among his immedi- 



History Taking 13 

ate family or associates, particularly among those with whom 
he has been living. State the reply to this question in the 
history whether it be negative or positive. 

IV. Previous History. — Write only the positive replies 
to the following questions, but be sure and do not omit any 
of the questions. When positive answers are obtained to ques- 
tions, ascertain time when the patient had this disease and 
express this time in days, weeks, months or years before fhe 
present illness. Avoid the use of dates in histories. They are 
confusing. 

1. General Diseases. — Ask specifically in regard to mea- 
sles, mumps, scarlet fever, diphtheria, smallpox, typhoid fever, 
malaria, pneumonia, ''inflammatory rheumatism," and menin- 
gitis. 

2. Diseases Pertaining to the Head and Throat. — 

(a) Frequent or severe attacks of headaches? Vertigo? 
Fainting spells? 

(b) Frequent or severe attacks of sore throat ■? Tonsillitis? 
Laryngitis ? Nose-bleed ? 

( c ) Sore ' mouth ? Eed tongue ? ' ' Salivation ? ' ' 

(d) Frequent or severe attacks of earache? Running 
ears ? 

(e) Bad eyes? 

3. Diseases Pertaining to the Chest. — 

Cough? Bronchitis? Shortness of breath on exertion? 
Hemoptysis? Loss of weight? Swelling of feet or legs? 
Night sweats ? Palpitation of the heart ? 

4. Diseases Pertaining to the Abdomen. — 

Vomiting attacks? Abdominal pains, cramps or colics? 

Indigestion? Eructations of gas? Heart burn? Hemateme- 

sis ? Constipation ? Diarrhea ? Visible blood in stools ? 
Jaundice ? 



14 Clinical and Laboratory Technic 

5. Diseases Pertaining to the Genito-Urinary System. 

(a) If a woman, ask in regard to menses, whether regular, 
profuse or painful. Also date of last menstrual period. Mis- 
carriages or abortions ? If so, time and number. 

(b) Polyuria? Nocturia? (State number of times per 
night.) Painful or difficult urination? Hematuria? 

(c) Gonorrhea? Chancre? Skin diseases? Swelling of 
testicles? Buboes? 

6. Diseases of the Nervous System. — 

Paralysis? Convulsions? Weakness of limbs? Paresthe- 
sias ? Burning, tingling, numbness ? Headaches ? Fainting 
spells ? Sleeplessness ? Vertigo ? 

7. Habits. — 

(a) Alcohol? (If used, state kind used most frequently 
and average amount used per day or week.) 

(b) Eating. — Rapid eating? Excess of carbohydrates? 
Heavy meat eater? 

(c) Coffee. In moderation? In excess? Tobacco (chew- 
ing, smoking) in moderation? In excess? 

(d) Drugs. — Morphin? Opium? Cocain? 

V. Present Illness- 
Describe chief or leading symptom first. Note its time of 
first appearance and its duration, then note other symptoms 
complained of. Avoid asking leading questions in eliciting 
symptoms. Do not use dates in describing time of onset of 
illness, but express time of onset in days, weeks or months 
before the present examination. Ask specifically in regard to 
symptoms referred to: 

(a) Head.— Nose bleed? Earache? Headache? Faint- 
ing? Sore throat? 

(b) CJiest. — Cough? Expectoration (amount and kind) ? 
Dyspnea? Pain? Hemoptysis? Palpitation? Orthopnea? 



History Taking 15 

(c) Abdomen. — Abdominal pain or cramps? Vomiting? 
Diarrhea? Constipation? Hematemesis? Melena? Piles? 
Pruritus ani? 

(d) Genitourinary. — Vaginal or urethral discharge? 
Polyuria? Oliguria? Dysuria? Hematuria? Painful or ir- 
regular menstruation (if woman) ? Last menstruation (if 
woman) ? 

(e) Fever? Night sweats? Loss of weight? 



PART II. 

PHYSICAL EXAMINATION. 

General Remarks. — 

In making a physical examination be sure to investigate 
thoroughly each of the points mentioned in the following 
outline. When, after such investigation, however, no abnor- 
mality is found in a particular system; the head and neck, 
for example, simply state "negative" under that heading. 

On the other hand, if any abnormality be found, describe 
it carefully and exactly ; when possible, using the terminology 
suggested in the outline which follows rather than coined 
terms., Moreover, if any organ such as the liver, heart, etc., be 
found enlarged, be sure to mention the size of this enlargement 
in centimeters, preferably; but if such measurement is not 
possible, in terms of inches, or, at least, finger-breadths. 

Do not try to hurry the examination. A good physical ex- 
amination should take from one-half to two or more hours for 
its completion, depending upon the training of the observer 
and upon the nature of the findings. 

Do not try to make a physical examination unless you can 
carry it out comfortably, in a quiet place, and with the clothes 
of the patient entirely removed from the chest and abdomen. 
Unless these precautions are taken at the outset of the exam- 
ination, an accurate physical examination cannot be made even 
by an expert. 

If in doubt as to any supposed abnormality in the examina- 
tion, write down what you think you hear, see, or feel, and 
then either confirm or refute it by a subsequent examination 
upon the following day. Do not fail to write down what you 
find since only by so committing yourself can accurate phys- 
ical diagnosis be learned. 

16 



Physical Examinati IT 

I. General Appearance. — 

1. Pallor? Jaundice? Cyanosis 1 Face flushed? 

2. Dull? Stupid? Comatose? Delirious? Bright? 

3. Emaciated? Well nourished ? Skin eruptions ? Edema? 

II. Head and Neck. — 

1. Deformities. — Tumors? Enlargement of thyroid ? Ede- 
ma of face or eyelids ? If a baby, is head large, rachitic, asym- 
metrical ? Are f ontanelles closed or open ? 

2. Eyes. — Exophthalmos? Arcus senilis 1 Conjunctivitis? 
Sclera jaundiced? Strabismus? 

3. Ears. — Discharge? Hearing good? 

4. Teeth and Gums. — Carious teeth? Deformed teeth 
( ' " Hutchinson ") ? Pyorrhea ? Bleeding gums ? Sordes ? 

5. Mouth and Throat. — 

(a) Tongue.— Palel Clean? Coated? Dry? Red? Moist? 
Fissured ? 

(b) Mouth. — Stomatitis? Mucus patches ? Koplik's spots ? 
Pharynx. — Tonsils enlarged ? Membrane? Inflamed? 

III. Nervous System. — 

(a) Pupils. — Are they equal ? Do they react to light and 
accommodation? (Argyle Robinson.) Is there any nystag- 
mus ? Strabismus ? Is the sight of both eyes apparently good ? 

(b) Reflexes. — Are any of the deep reflexes exaggerated 
or diminished ? I Knee jerks ; Triceps ; Biceps. ) Is Babinski's 
sign present? Kernig's sign? Romberg's sign? Ankle 
clon 

(c) Sensations. — Are there any areas of anesthesia to 
pain ? touch ? heat ? cold ? Is the sense of taste and smell in- 
tact? Are there any sensations of paresthesia? I'Xumbness. 
tingling, bnrning 



18 Clinical and Laboratory Teclinic 

(d) Coordination. — Can patient use his. hands and feet 
well with the eyes closed ? ( Can he touch his nose with the in- 
dex finger? Place his right heel upon his left knee? Recog- 
nize objects by their feel alone, such as coins, a bunch of 
keys, a knife?) Is there any tremor present, either of the 
fingers, tongue, or extremities? If so, is it coarse or fine? 

(e) Speech. — Is the articulation good? 

(f) Gait. — Unsteadiness or incoordination? Spastic? 

IV. Chest.— 

1. Inspection. — Asymmetrical? Respirations rapid? Res- 
pirations labored? Movements unequal on two sides? Ab- 
normal pulsations ? Tumors ? 

2. Palpation.— Tactile fremitus? Thrills? Tender points ? 

3. Percussion. — Dullness? Flatness? Cracked pot sound ? 
upper and lower borders of lungs in normal location? Up- 
per border of liver in normal position? Is percussion note 
clear throughout? 

4. Auscultation. — 

(a) Tubular breathing? If tubular breathing is present 
state exact location, using following landmarks: Front. — 
Right or left supra- or infra-clavicular fossa. Right or left, 
upper or lower axilla. Back. — Right or left apex in back. 
Right or left inter-scapular area. Right or left lower back. 

(b) Amphoric breathing? If so, state location as for tubu- 
lar breathing. 

(c) Broncho-vesicular breathing? If so, state location, as 
for tubular breathing. 

(d) Suppressed {distant), or absent breath sounds? If 
so, state location. 

(e) Breath sounds vesicular throughout? 

(f) Friction rubs or Rales? If rales are present, state 
kind (whether coarse, medium, or fine moist rales; or whether 
sonorous, sibilant, or fine dry rales) . State exact location of 



Physical Examination 19 

friction rub, or rales, using same landmarks as for tubular 
breathing, also state whether they are audible at end of in- 
spiration, expiration or both. 

(g) Are voice sounds well transmitted throughout? Di- 
minished? Increased? (If so, where?) 

V. Cardiovascular System. — 

1 . Inspection. — 

Position of apex beat? What interspace? Is it inside or 
outside the mml. ? If outside how many finger-breadths? 
[Abbreviations may be used as follows: Instead of writing 
"Apex beat in sixth intercostal space, three finger-breadths 
outside mammary line," write, P.M.I, (point of maximum im- 
pulse) in 6th i.s. 3 f.b. outside mml. ; P.M.I. 1 f.b. inside mml. 
in 5th i.s., etc.] 

2. Palpation. — 

■ (a) Confirm position of P.M.I. 

(b) Thrill? (Diastolic? Systolic? Presystolic?) 

(c) Diastolic or systolic shock? 

3. Percussion. — 

(a) Right and left borders of cardiac dullness. Express 
in centimeters to right or left of mid-sternal line, mentioning 
interspace in which percussion was made. [Abbreviations may 
be used as follows: L.C.D. (left cardiac dullness) 12 cm. to 
left of M.S.I, in 6th ijs. R.C.D. 4 cm. to right of M.S.I, in 3rd 
i.s., etc.] 

(b) Dullness under manubrium? 

4. Auscultation. — 

(a) Heart sounds. — Is either sound accentuated? (If so, 
which?) Either sound muffled? Arrhythmia? Extrasys- 
toles? Reduplication of sounds? 

(b) Murmurs. — Kind? (Soft, blowing, rough, musical?) 
Time? (Whether systolic, presystolic, or diastolic?) Where 



20 Clinical and Laboratory Technic 

is the murmur of maximum intensity? (Base, apex, or to left 
of sternum?) Transmission of murmur? (Outwards from 
apex? Into vessels of neck?) 

(c) Pericardial friction rub? 

5. Pulse. — 

Are both radial pulses synchronous and equal? Are ar- 
teries sclerotic? (Radial? Temporal?) Is pulse regular in 
force and rhythm ? State rate of pulse per minute. 

VI. Abdomen. — 

1. Inspection. — 

Full ? Distended ? Scaphoid ? Abnormal masses ? Abnor- 
mal pulsations? Rose spots? Eruptions? 

2. Palpation. — 

Tenderness? Rigidity? (If present describe exact loca- 
tion using the following terms in describing position : Right 
and left hypochondrium ; right and left iliac fossa; epigas- 
trium ; above or below umbilicus ( stating distance above or be- 
low in finger-breadths). Pulsations? Masses? Is edge of 
spleen palpable ? Is lower border of liver palpable ? Are kid- 
neys palpable ? 

3. Percussion. — 

(a) Lower border of liver. If below costal margin, state 
location of the lower border in the terms of centimeters or 
finger-breadths below costal margin in the mammary line. 
(Abbreviations may be used here, as L.D. 4 f.b. below cm. in 
mml.) Similarly, if lower border is above the costal margin 
in the mammary line, so state. 

(b) Is splenic dullness apparently increased? 

( c) Tympanitic-abdomen ? 

(d) Movable dullness? (Ascites?) 

VII. Genito-Urinary. — 

Chancre? Scars on genitals? Urethral discharge? Orchi- 
tis? Buboes? 



i 



Physical Examination 21 

VIII. Rectum.— 

Piles ? Fissure ? Prolapse ? 

IX. Spine and Extremities. — 

1. Spine. — Curvature? Tender points? 

2. Extremities. — Deformities? Tumors? Ulcers? Pig- 
mented areas? Clubbed fingers? Eruptions, sunburn, blis- 
ters, etc., on legs, hands or arms? (Pellagra?). Tenderness 
of bones or muscles? Glandular enlargements (epitrochlears, 
axillary, posterior and anterior cervical, inguinal) ? 



PART III. 

COMMON LABORATORY METHODS USED IN 
DIAGNOSIS. 



EXAMINATION OF THE URINE. 

Quantity. — The normal quantity for an adult, in 24 
hours, in a moderately warm climate varies between 900 and 
1500 c.c., depending upon the temperature, water intake, etc. 
(Amount apt to be increased in diabetes mellitus, chronic ne- 
phritis and diabetes insipidus.) 

Color. — In describing color of urines, certain standard 
terms should be used for the sake of uniformity. The terms 
commonly used for such description are: 1, pale yellow; 2, 
yellow; 3, amber; 4, brown; 5, dark brown. 

Reaction. — Acid 1 Alkaline ? . Amphoteric 1 

Specific Gravity. — The normal specific gravity varies from 
1015 to 1025, depending upon temperature, water intake, 
diet, etc. (In case not enough urine is on hand to fill the 
specific gravity cylinder, the urine may be diluted with a 
known volume of water. The formula for correction being: 
Sp.g. equals 1000 plus ab, in which b is the dilution, and a is 
the last two figures of the specific gravity found. For in- 
stance, if the urine was diluted three times (with twice its 
volume, or two parts, of water) and the reading of the specific 
gravity were 1008, the true sp.g. would be 1000 plus 3 mul- 
tiplied by 8, which is 1024. If the urine were diluted to only 
twice its volume the gravity would be 1000 plus 2x8 or 1016.) 



Sediment. — Granular 



Flocculent? Mucous threads? 

22 



Examination of tlie Urine 23 

Albumin. — Use any of the following tests: 

1. Robert's Test. — 
Necessary Reagents. — 

Concentrated nitric acid 1 part. 

Saturated solution of magnesium sulphate .... 5 parts. 

Place a small quantity of urine over Robert's solution. If 
albumin is present a cloudy ring will form at the point of 
contact of the two fluids. 

2. Heat and Acetic Acid Test. — 
Necessary Reagents. — 

5% acetic acid. 

Fill test tube one-half full of urine- Heat the upper portion 
of the urine in a medium-sized flame until it begins to boil. 
If albumin is present a cloudiness appears in the heated por- 
tion of the urine. A slight cloud is best distinguished by 
holding the tube against a dark background and comparing 
the heated with the unheated portion of urine. After heating 
the urine and looking for a cloud, add four or five drops of 
5% acetic acid. Heat upper portion of the urine again. If 
cloudiness was caused by albumin it will remain, or become 
even more distinct. If it was caused by phosphates, however, 
it will disappear as soon as the acid is added. 

3. Heat and Nitric Acid Test. — 
Necessary Reagents. — 

Concentrated nitric acid. 

Done in exactly the same way as the heat and acetic test, 
except that, after boiling, a few drops of strong nitric acid 
are added instead of dilute acetic acid. In this test, also, 
phosphates are precipitated by heating, which disappear, how- 
ever, as soon as the acid is added, while the cloud caused by 
albumin remains. 



24 Clinical and Laboratory Teclinic 

4. Quantitative Albumin. — 

As a rule the quantity of albumin present in a given speci- 
men of urine may be estimated with sufficient accuracy for 
practical purposes, by noting the density of the albuminous 
cloud produced during any of the preceding tests ; this quan- 



— R 



— U 



W 

Fig. 1.— Esbach albuminometer. 

tity being expressed as: '"a trace;" "a moderate cloud;" "a 
heavy precipitate." 

If, however, one is examining a 24-hour specimen, or de- 
sires to express the quantity of albumin with scientific accu- 
racy it becomes necessary to use the Esbach albuminometer. 
This is done as folloAvs: 

Necessary Apparatus. — 
Esbach albuminometer. 



Examination of tlie Urine 25 

Necessary Reagents. — 

Esbach's Solution. 

Picric acid 10 gms. 

Citric acid 20 gms. 

Distilled water q. s. ad 1000 c.c. 

In carrying out this test the tube is filled to the mark "U" 
with the urine to be tested, then to the mark "R" with Es- 
bach 's solution. After carefully mixing by gentle inversion 
of the tightly corked tube for ten or more times, the whole is 
allowed to stand at room temperature for 24 hours. 

At the end of this time, the amount of albumin present in 
the urine, expressed in grams per liter, may be read directly 
off from the tube. 

Bence- Jones Bodies. — 

The appearance of a cloud in the urine on the addition of 
acid, which disappears on boiling and reappears on cooling, 
signifies the presence of albumoses, or "Bence- Jones bodies. 



i •> 



Sugar. — 

1. Fehling's Test (Qualitative). — 
Necessary Reagents. — 

Solution A. — "Fehling's Copper." 

Copper sulphate 34.65 gms. 

Distilled water q. s. ad 1000 c.c. 

Solution B. — ' ' Fehling 's A Ikaline. ' ' 

Rochelle salt 173 gms. 

Sodium hydrate 125 gms. 

Distilled water q. s. ad 1000 c.c. 

Mix equal parts of solutions A and B (about two c.c. of 
each), and boil. Now add urine slowly, drop by drop, up to 
the amount of one of the solutions used (2 c.c). Boil after 
each fresh addition of urine. 

If sugar is present, a yellow precipitate of copper oxide 
will form at once. Unless a definite yellow precipitate forms 



26 Clinical and Laboratory Tecknic 

promptly, the test is not to be considered positive. In nor- 
mal urines, a slight change in color will sometimes take place 
when the urine is treated in this way. Also, urines to which 
certain preservatives have been added, such as chloroform, 
will give a very definite color change. In none of these urines, 
however, will the typical yellow heavy precipitate of copper 
oxide be noted. This test is also given by other substances 
than glucose, as: lactose, pentose, levulose, and glycuronic 
acid. 

2. Benedict's Test. — 
Necessary Reagents.-^- 

Benedict's Solution. 

Copper sulphate 17.3 gms. 

Sodium citrate 173 gms. 

Sodium carbonate . . 200 gms. 

Distilled water '. . . q. s. ad 1000 c.c. 

Dissolve copper sulphate separately in 100 c.c. of distilled 
water before adding to solution. 

To carry out this test, fill a test tube one-third full (5 c.c.) 
of Benedict's solution and bring to boil. Add urine slowly 
as in preceding test, boiling after each addition. (Not more 
than eight drops of urine should be added to 5 c.c. of the 
solution.) 

If glucose is present, the typical yellow precipitate of cop- 
per oxide will form promptly. This test is also given by 
lactose, levulose and pentose, but not by glycuronic acid. 

3. Fermentation Test. — 
Necessary Reagents. — 

Fresh yeast. 
Fermentation tube. 

A piece of fresh yeast about the size of a pea is added to 
a sufficient amount of urine to fill a fermentation tube. The 
urine is then shaken gently and poured into the fermentation 
tube which is allowed to stand at room temperature for sev- 



Examination of the TJrim 27 

era! hours. If glucose is present, gas will collect in the closed 
upper arm of the fermentation tube. (A control tube should 
be used to which no yeast has been added.) 

This test is given by no sugar which is apt to be found in 
the urine, except glucose, so that a positive fermentation test 
is fairly good evidence of true glycosuria if confirmed by a 
reduction of Fehling's or of Benedict's solution. (Levulose 
is the only other sugar found in the urine which ferments 
yeast and reduces copper sulphate, but levulosuria is so rare 
as to be practically negligible.) If a reducing body is found 
in the urine which does not ferment with yeast, it is probably 
lactose, which is especially common in lactating or pregnant 
women. 

Quantitative Tests for Sugar. — 

1. Benedict's Test. — 
Necessary Apparatus. — 

1 Burette with stand. 

1 Porcelain dish. 

1 25 c.c. graduated cylinder. 

Necessary Reagents. — 

" Benedict's Quantitative Solution. 

Copper sulphate IS gins. 

Sodium carbonate 200 gms. 

Sodium citrate 200 gms. 

Potassium sulphocyanate 125 gms. 

5% solution potassium ferrocyanide 5 c.c. 

Distilled water q. s. ad 1000 c.c. 

Dissolve copper sulphate separately in 100 c.c. of dis- 
tilled water before adding to rest of solution. If made 
properly, 25 c.c. of this solution are reduced by 50 milli- 
grams of glucose. 

To determine the quantity of sugar in a given urine, meas- 
ure out exactly 25 c.c. of Benedict's quantitative solution and 
bring to a brisk boil in a white porcelain dish. After solution 
has begun to boil briskly add the urine to be tested, (from a 
burette), until the color of the solution begins to fade. As 



28 



Clinical and Laboratory Teclinic 



soon as a slight change in color is noted begin to add the urine 
very slowly, drop by drop, boiling well after each addition. 
When the blue color has entirely disappeared, the solution 
having taken on a pure white color, note the exact amount 
of urine which has been used to reduce the solution. Know- 
ing that this amount of urine contains 50 milligrams of glu- 
cose (the solution is so made that 25 c.c. of Benedict's is re- 
duced by 50 mgs. of glucose), it is easy to determine how many 
grams or fractions of a gram of glucose will be contained in 




Fig. 2.— Einhorn saccharometer. 



1 c.c. of urine. Multiplying this amount by 100 will give us 
the number of grams in 100 c.c. of urine. This number of 
grams per 100 c.c. of urine will represent the percentage of 
sugar present. In doing this test, to prevent boiling over of 
the solution, it is well to add 10 to 20 grams of sodium car- 
bonate or a small amount of pumice stone to the solution, be- 
fore boiling. 

2. Fermentation Test. — 
Necessary Reagents. — 

One Einhorn saccharometer. 
Absolutely fresh yeast. 



Examination of the Urine 29 

If the specific gravity of the urine is as high as 1030 or 
over, dilute the urine four or five times. Fill the sacchari- 
meter with the urine, to which a piece of yeast has been added, 
as in the qualitative fermentation test. Keep in a warm place 
(room) for 24 hours. At the end of this time read off directly 
from the graduated scale on the closed arm of the tube the 
percentage of sugar which was present. (The amount of car- 
bonic acid gas formed is used as a measure of the amount 
of sugar present.) In doing this test, it is well to use a con- 
trol tube containing a known percentage of glucose before ac- 
cepting this reading as absolutely accurate. The test is ex- 
tremely simple, however, and fairly satisfactory for small 
amounts of sugar. 

Acetone. — 

1. Legal 's Test. — 
Necessary Reagents. — 

Sodium nitroprusside crystals. 
Strong solution of sod. hydroxide. 
Glacial acetic acid. 

To one-sixth of a test tube full of urine (2 or 3 c.c.) add 
one crystal of sodium nitroprusside. Make solution strongly 
alkaline with sodium hydroxide. Shake. Add a few drops of 
glacial acetic acid. If acetone is present, a deep purple color 
will make its appearance on the addition of the acetic acid. 

2. Gunning's Test. — 
Necessary Reagents. — 

Tincture of iodine or Lugol 's solution. 
Ammonia. 

To one-third of a test tube full of urine, using preferably 
urinary distillate, add a few drops of tincture of iodine or 
of Lugol's solution. Then add ammonia until a deep black 
precipitate forms. If acetone is present, the black precipitate 
will, on standing, disappear, leaving a yellow sediment of iodo- 



30 Clinical and Laboratory Teclinic 

form, which may be recognized by its color, odor, and char- 
acteristic six-sided crystals. 

Gunning's test is, as a rule, used only when confirmatory 
evidence of the presence of acetone is needed. 

Diacetic Acid. — 

Gerhardt's Test. — 
Necessary Reagents. — 

A strong aqueous solution of ferric chloride. 

To a test tube one-third full of urine add the ferric chloride 
solution in excess. If a deep, bordeaux red color makes its 
appearance, the test is positive. If a heavy precipitate oc- 
curs on the addition of the ferric chloride solution, filter the 
urine and add more ferric chloride solution to the filtrate. 

This test is given by several other bodies than diacetic acid, 
and is especially apt to be misleading in urines from patients 
who have been taking any of the salicylic acid compounds, 
such as aspirin, diuretin, phenacetin, salol, and others. 

In order to rule out these bodies, which are non-volatile, 
boil the urine before adding the ferric chloride solution. If 
diacetic acid, which acid is volatile, was the substance which 
caused the color reaction, the reaction will now be absent in 
the boiled urine, while if it was any of the bodies mentioned 
it will be unaffected. 

Beta-Oxybutyric Acid. — 

This test is not easily carried out and, since the presence 
of diacetic acid usually means the presence of (3 oxybutyric 
also, no test for the latter substance is done, as a rule. 

Diazo Reactions. — 

Necessary Reagents. — 

Solution A. 

Sulphanilie acid 1 gm. 

Hydrochloric acid 50 c.c. 

Distilled water 1000 c.c. 



Examination of tine Urine 31 

Solution B. 

Sodium nitrite 1 gm. 

Distilled water 200 c.c. 

Keep solutions separate, in well corked, dark bottles. 

To 50 parts of solution A, add one part of solution B. (Five 
c.c. of A, plus two drops of B.) Add an equal amount of 
urine. Shake and add quickly, -2 or 3 c.c. of ammonium hy- 
droxide. If reaction is positive, a deep cherry red or carmine 
color will make its appearance. On shaking, the foam will 
be a distinct rose red color. Normal urines will give a brown- 
ish-red color with a yellowish-pink foam, so that a definite 
deep red color must be obtained in the fluid and at least a 
distinct rose red in the foam before the reaction is considered 
positive. 

Indican.— 

Necessary Reagents.— 

A. Lead acetate, 20% solution. 

B. Obermayer's reagent: 

Concentrated hydrochloric acid 100 c.c. 

Ferric chloride 1 gm. 

To 20 c.c. of urine, add 4 c.c. of 20% lead acetate. Shake. 
Filter. To one-third test tube full of filtrate, add an equal 
amount of Obermayer's reagent. Shake well. Add one c.c. 
of chloroform and shake again. If indican is present in ab- 
normal quantity the chloroform will assume a deep blue color. 
A blue color ranging from sky blue to bluish-black denotes an 
increase in indican above normal. 

Bile.— 

1. Shake urine well in a test tube. With the presence 
of bile, the foam will take a deep yellow color. 

2. Rosenbach's Test. — 

Filter 20 to 40 c.c. of urine (a large test tube full), through 
one small filter paper several times. Dry this paper with 
another piece of filter paper. Add one drop of yellow nitric 
acid to the filter paper, through which the urine has been fil- 



32 Clinical and Laboratory Teclinic 

tered. If bile is present a stratum of colors will form around 
this drop of acid, the colors ranging from green without to 
blue, violet and red within. This test is very delicate. 

Blood.— 

1. Guaiac Test. — 
Necessary Reagents. — 

Fresh tincture of guaiac* 

Fresh hydrogen peroxide. 

Glacial acetic acid. 

*Made by adding a small particle of gum guaiac to a few c.c. of 95% alcohol 
and shaking well. 

To one-half c.c. of fresh tincture of guaiac, add fresh hy- 
drogen peroxide until a distinct milky color is obtained. Mix 
well and add an equal quantity of urine, to which a few drops 
of glacial acetic acid have been previously added. If blood 
is present, a dark green color will form promptly. The reac- 
tion should be read within five minutes. 

2: Benzidine Test. — 
Necessary Reagents. — 

Fresh tincture of benzidine.* 
Fresh hydrogen peroxide. 
Glacial acetic acid. 

*Add a small quantity of powdered benzidine to a small amount of 95% alcohol 
and shake well. 

Mix equal parts of: (a) Fresh tincture of benzidine; (b) 
Hydrogen peroxide; and (c) Urine, which has been made 
strongly acid with a few drops of glacial acetic acid. If blood 
is present, a bluish-green color will be produced. 

3. Meyer's Test. — 

Necessary Reagents.— 

Phenolphthalein 4 gms. 

Potassium hydroxide '. . . 35 gms. 

Distilled water 200 c.c. 

Mix and allow to stand 3 minutes, then add pure zinc 
dust, 20 gms., and boil until decolorized. Keep in a dark, 
well stoppered bottle, along with the zinc dust. 



Examination of ilu Urine 



33 



Place equal parts of this solution and the solution to be 
tested in a test tube. Shake and add a few drops of hydrogen 
peroxide. If test is positive for blood, a pink color will form 
upon the addition of the hydrogen peroxide. 




Fig. 3. — Doremus-Hinds ureometer. 



Urea. (Quantitative estimation. )- 



This test is of no value unless a carefully measured 24-hour 
specimen of urine is used. 

Doremus Method. — 
Xecessary Apparatus. — 
Doremus ureometer. 



Necessary Reagents. 



Solution A. 

Sodium Irydroxide 100 gins. 

Distilled water 250 c.c. 



34 Clinical and Laboratory Technic 

Solution B. 

Bromine 25 gms. 

Potassium bromide 25 gms. 

Distilled water 200 c.c. 

The test is carried out by mixing equal quantities of solu- 
tions A and B (which, if kept separate, are fairly stable). 
The ureometer tube is then filled with this mixture, care being 
taken to see that the closed arm of the tube contains no air. 
Exactly one cubic centimeter of the urine to be tested is now 
allowed to flow into the upper, closed arm, of the ureometer 
and the tube is allowed to stand for several minutes (15 min- 
utes), at the end of which time the amount of gas which has 
collected in the upper part of the closed tube is noted. The 
graduations on this closed arm represent the number of milli- 
grams of urea present in the one cubic centimeter of urine 
which was added to the solution. Knowing the amount of 
urea in one cubic centimeter of urine, in order to obtain the 
total quantity excreted in 24 hours, it is simply necessary to 
multiply this amount by the number of cubic centimeters of 
urine excreted. The normal excretion of urea in an adult is 
from 20 to 40 gms. in 24 hours, the amount depending largely 
upon the diet. 

Microscopic Examination. — 

Necessary Apparatus. — 

Centrifuge. 
Centrifuge tubes. 
Small pipette. 

Before making a microscopical examination of the urine, it 
is advisable to centrifuge for a short time at moderate speed 
in order to throw down the solid elements. A small amount 
of this sediment is now removed by means of a small pipette 
and placed upon a slide. Apply cover glass and examine first 
with low power lens, then with the high power. Do not use 
oil immersion. 



Examination of tlie Urine 



35 





Fig. 4. — Common epithelial cells met with in the urine. A, Squamous cell of 
vagina and urethra; B, Caudate cells from pelvis of kidney, ureter, and bladder; 
C, Cylindrical cell from urethra; D J Cuboidal, or so-called "renal cells." 




Fig. 5. — Finely granular casts. 




Fig. 6. — Coarsely granular casts. 




Fig. 7. — Hyaline cast. 




Fig. 9. — Pus casts. 




Illl 
Fig. 11. — Epithelial casts. 



Fig. 8. — Waxy casts. 




Fig. 10. — Blood casts. 



/ X/ 



1 



SSI 



\ Hm^m 



& 




Fig. 12. — Mucous threads. 



36 



Clinical and Laboratory Technic 



1. Casts. — 

Epithelial? Pusl 

granular ? Hyaline 



Blood? Coarsely granular? Finely 
Waxy? 



2. Leucocytes. — (Few? Many?) 

3. Eed Blood Cells. — 

If in doubt as to whether cells are red blood cells or leu- 
cocytes, add two or three drops of 5% acetic acid to slide and 
mix well. Red blood cells will disappear, white will remain. 




Fig. 13. — Common crystals found in urinary sediments. A, Uric acid; B, Am- 
monium urate; C, Calcium oxalate; D, Triple phosphate; B, Calcium carbonate. 

4. Crystals. — (Kind?) (Alkaline urines are apt to con- 
tain either phosphates or carbonates. Acid urines are apt to 
contain urates or uric acid crystals.) 

5. Tubercle Bacilli. — 

It is desirable to use only catheterized specimens of urine 
for this test, in order to avoid confusion with the smegma 
bacillus, which is a normal inhabitant of the lower urinary 
passages. 

(a) Fill a thoroughly clean centrifuge tube (cleansed with 
concentrated hydrochloric or sulphuric acid), with the sus- 
pected urine. Centrifuge for a short time (one minute) at 
moderate speed in order to throw down the heavier sediment 
such as epithelial cells, casts, pus cells, and crystals. Since 
the tubercle bacilli are relatively much lighter than the sedi- 
ments, they do not come down with them, but are only thrown 
down after prolonged and rapid centrifuging. After cen- 



Examination of Hie Urine 37 

trifuging one minute at moderate speed, pour the supernatant 
fluid into another clean centrifuge tube and centrifuge as 
rapidly as possible for twenty minutes (1500-3000 R.P.M.). 
Pour off supernatant fluid and refill centrifuge tube with dis- 
tilled water. Mix contents and recentrifuge at same speed 
for twenty minutes. Pour off supernatant fluid, spread sedi- 
ment on slide, dry and fix with heat. 

(b) Stain for tubercle bacilli as described under sputum. 
If acid-fast organisms are found in a non-catlieterized speci- 
men of urine, repeat tlie test ivitli a catlieterized specimen 
before making a diagnosis of tuberculosis. 

6. Gonococci. — 

Xecessary Reagents. — 

Lugol's solution.-' (Gram's solution.) 
Carbol-gentian violet.* 
95% alcohol. 
2% safranine. 

*For directions as to making these stains, see appendix. 

(a) Smear slide thinly with suspected material, dry and 
fix by heat. 

(b) Stain for two minutes with carbol-gentian violet.* 

(c) Cover with Lugol's solution for one minute (without 
washing). 

(d) Wash with 95% alcohol until no more blue color comes 
out (30 seconds is sufficient with thin smear). 

(e) Wash in water. 

(f) Counter-stain with 2% safranine* or with dilute car- 
bol-fuchsin (1 to 10) for one minute without heat. 

(g) Wash in water, dry and examine with oil immersion. 
Gonococci show as reddish, biscuit-shaped, intracellular diplo- 
cocci. 



'For directions as to making these stains, see appendix. 



38 Clinical and Laboratory Technic 

EXAMINATION OF THE BLOOD. 

I. White Counting". — 

Necessary Apparatus. — 

Blood sticker. 

Mixing pipette for white cells. 

Counting chamber with cover-glass. 

Necessary Reagents. — 

Fresh 1% acetic acid. 

Alcohol. 

Ether. 

Be sure that the counting pipette is both clean and dry be- 
fore attempting to begin the count. If dirty or if it contains 
moisture, as shown by the small glass bead clinging to the 
walls of the pipette, wash successively with water, alcohol and 
ether until perfectly dry. After puncturing the finger or ear 
of the patient, being sure to puncture deep enough to obtain 
a fairly free now of blood, draw up blood quickly to the mark 
"0.5," in the white pipette. It is absolutely necessary, in or- 
der to obtain an accurate count, that this column of blood be 
drawn exactly to this 0.5 mark and not above or below it, 
since a variation of as small a fraction as a millimeter will 
cause a marked variation in the white count. (Only after 
considerable practice can an accurate column of blood be ob- 
tained.) As soon as the blood has been drawn to the proper 
mark, quickly draw up 1% acetic acid to the mark "11" on 
the pipette, rotating the pipette gently while drawing up the 
solution. As soon as the chamber is filled to the mark "11," 
shake the pipette well, holding the ends between the fingers 
to prevent any fluid escaping. Allow to stand for a few min- 
utes before beginning the count. 

When ready to count, clean counting chamber and cover- 
glass (never use alcoliol on the- counting chamber since it dis- 
solves the cement) , and dry well, being careful that no small 
particles of lint or dust are left on the surfaces which are to 
be approximated. 



Examination of the Blood 



39 



Express two or three drops of fluid from the pipette in or- 
der to obtain the well mixed portion for counting. Place a 
small drop of the remaining mixture upon the plateau of the 
counting chamber. (The size of this drop is very important 
and can only be learned by practice. It must not be large 
enough to run off of the plateau into the surrounding moat 
when the cover-glass is superimposed, yet it must be large 
enough to cover the entire plateau when the cover-glass is 
placed over it.) "When this small drop has been expressed 
upon the plateau, superimpose the cover-glass, and, being sure 
that the drop is neither too large nor too small, next make 




Fig. 14. — "Thoma" counting chamber. 

sure that the cover-glass is well approximated. This can be 
determined by the finding of "Newton's rings." 

A perfect drop being now obtained and the cover-glass well 
approximated one is ready to begin the white count, which 
is done by counting all white cells contained upon the square 
millimeter which is ruled off on the plateau. The low power 
of the microscope is best used for this count, although the high 
power may be used. Now make a new drop from the pipette 
in the same way and count the number of white cells on an- 
other square millimeter. The cells on four square millimeters 
are counted in this way, preferably using a- fresh drop for 
each square millimeter, and the average number of cells con- 
tained on one square millimeter is thus estimated. 

In order to ascertain the actual number of white cells con- 
tained in one cubic millimeter of the mixture it is necessary 
to know the thickness of the blood which has just been counted. 



40 Clinical and Laboratory Technic 

All counting chambers are so adjusted that the thickness of 
the drop, when the cover-glass is superimposed, is exactly one- 
tenth of a millimeter. The number of cells contained in one 
cubic millimeter of the mixture will therefore be obtained by 
multiplying the result of the count by ten. 

Knowing, now, the dilution of the blood which was 1 to 
20 ; in order to obtain the actual number of cells in one cubic 
millimeter of whole blood we again multiply by this dilution 
(20), which gives us the total number of white cells in one 
cubic millimeter of whole blood. 

For example, if, after counting four squares, we found an 
average of 31 cells on one square millimeter, in order to ob- 
tain the number of white cells in the whole blood, we simply 
multiply thirty-one by ten and by twenty (31X10X20 = 
6200), which would give us a white blood count of 6200 to 
the cubic millimeter. 

II. Red Counting". — 

Necessary Apparatus. — 

Counting chamber, with cover-glass. 
Mixing pipette for red cells. 
Blood sticker. 

Necessary Reagents. — 

Hayem's Solution: 

Distilled water .200 c.c. 

Sodium chloride 1 gm. 

Sodium sulphate 5 gms. 

Mercuric chloride 0.5 gm. 

Alcohol (95%). 
Ether. 

See that mixing pipette is clean and dry as in counting 
whites. Draw up blood exactly to 0.5 mark, in the pipette, 
being sure that column does not extend either below or above 
this mark. As soon as the proper amount of blood is ob- 
tained, quickly draw up Hayem's solution to the mark 101 
in the pipette. Shake well. After making sure that the cov- 
er-glass and counting chamber are perfectly clean and free 



Examination of the Blood 41 

from lint and dust, make a drop of exactly the proper size, 
as in white counting, and apply the cover-glass. If perfect 
apposition of the cover-glass is obtained (Newton's rings), 
count the number of red cells contained on one-sixteenth of a 
square millimeter (instead of on one whole square millimeter 
as in the white count). Make four such counts on different 
parts of the same preparation, then clean counting chamber 
and cover slip, make another drop, and count four more one- 
sixteenth square millimeters in the same way. Now add up 
the total of the eight one-sixteenth square millimeters and ob- 
tain the average number of cells contained on one-sixteenth 
of a square millimeter. Knowing that this number of cells is 
contained on one-sixteenth of a square millimeter, in order 
to obtain the number of cells over an area of one whole square 
millimeter, we multiply this figure by sixteen. 

Knowing that the drop is one-tenth of a millimeter in thick- 
ness, in order to obtain the number of cells in one whole cubic 
millimeter of the mixture, we simply multiply this figure by 
ten. Having done this, we now multiply by the dilution of 
the blood (200), which gives us the number of red cells con- 
tained in one cubic millimeter of whole blood. 

For example,. if the average number of cells, obtained after 
a count of eight one-sixteenths of a square millimeter, was 
150, the number of cells contained in one cubic millimeter of 
the whole blood would be 150X16X10X200 = 4,800,000 cells 
per cubic millimeter. 

III. Hemoglobin. — 

Necessary Apparatus. — 

Sahli hemometer. 
Blood sticker. 

Necessary Reagents. — 

Tenth normal hydrochloric acid. 

Distilled water. 

Alcohol. 

Ether. 



42 Clinical and Laboratory Technic 

See that apparatus is clean and dry. 

Place the tenth normal hydrochloric acid in the graduated 
mixing tube up to the mark 10. Draw up a column of the 
blood to be tested to the mark 20 in the pipette. Express this 
blood quickly into the graduated mixing tube, mix well and 
allow to stand for a few (five) minutes. Fill the graduated 




Fig. 15. — Sahli hemometer. 

mixing tube carefully and slowly with distilled water until 
the color of the mixture exactly matches that of the solution 
in the standard tube. When the colors are matched read the 
percentage of hemoglobin directly from the scale on the grad- 
uated mixing tube. 



Examination of the Blood 43 

IV. Differential Counting. — 

Necessary Apparatus. — 

Clean slides. 
Blood sticker. 

Necessary Reagents. — 

Wright's stain.* 
Distilled water. 
Blotting paper. 

*For directions as to making stains see appendix. 

Clean two slides in running water. (Avoid the use of al- 
cohol in cleaning.) Dry thoroughly. Do not touch the sur- 
faces of the slides with the fingers after cleaning. Make a 
smear of the blood to be counted upon each of these slides. 
Be sure that the smear is very thin. Dry in the air. Cover 
with Wright's stain for 30 seconds., Add distilled water care- 
fully but quickly until a slight metallic scum makes its ap- 
pearance (usually about two parts of distilled water to one 
of stain are sufficient). Allow mixture to stand for three 
minutes. Wash off stain in running water (preferably dis- 
tilled), holding slide in a horizontal position while washing 
in order to prevent deposits of sediment. Blot promptly and 
examine under oil immersion. Count 200 white cells in mak- 
ing a differential. In making a differential always examine 
red cells carefully for malarial organisms, GTrawitz' granules, 
poikilocytosis, anisocytosis and nucleated red cells. 

V. Classification of Leucocytes. — 

The following classification is used in this clinic : 

I. Polymorphonuclear Neutrophiles. — 

Size averages about twice that of a red blood cell; varies 
somewhat, however. Nucleus is polymorphous and takes a 
light blue stain. Protoplasm is granular, containing numer- 
ous fine granules, which take a lilac or light pink stain. 

II. Polymorphonuclear Eosinophiles. (Eosinophile.) 
Size the same as that of the neutrophile. Nucleus polymor- 



44 Clinical and Laboratory Technic 

phous, and takes a light blue stain. Protoplasm is granular 
but contains very coarse granules as compared with, the neutro- 
phil, the granules taking a deep brick-red color. 

III. Polymorphonuclear Basophile. (Basophile.) — 

Size the same as that of a neutrophile or eosinophile. Neu- 
cleus polymorphous, taking a light blue stain. Protoplasm 
contains from a few to numerous coarse dark blue granules. 

IV. Lymphocyte. — 

Size averages about that of a red blood cell; varies, how- 
ever, between that of a red cell and that of a polymorpho- 
nuclear cell. Nucleus round or oval, but often contains an 
indentation in one side. Nucleus takes a deep blue stain. 
The nucleus is large as compared with the protoplasm of the 
cell, occupying the greater part of the cell as a rule. The 
protoplasm, which is often present as only a thin ring sur- 
rounding the nucleus takes a light blue stain and is non- 
granular. Occasionally, however, one or two very coarse blue 
granules are seen in the protoplasm of lymphocytes, the so- 
called, "azurophilic granules." The lymphocytes approach- 
ing the size of polymorphonuclear cells are sometimes called 
large lymphocytes as distinguished from the smaller and more 
common lymphocytes which approach more the size of the red 
cells. 

V. Large Mononuclears. — 

These cells are the largest found in the normal blood, aver- 
aging from about the size of a polymorphonuclear leucocyte 
to several times that size. The nucleus is relatively small as 
compared with the large amount of protoplasm. The nucleus 
is round or oval and is usually situated towards one side of 
the protoplasm. It takes a fairly deep blue stain. The proto- 
plasm of the large mononuclear is its most characteristic fea- 
ture, being relatively large in amount, taking a very light blue 
stain and being very delicate, so delicate in fact that it is 
very easily distorted during the process of staining, the edges 
being often rolled up upon themselves or torn off. 



Examination of the Blood 45 

VI. Transitioxals. — 

The transitional cell averages in size about that of the 
polymorphonuclear. The nucleus is kidney- or bean-shaped, 
having an indentation which is much more marked than that 
which is occasionally found in the nuclei of the lymphocytes. 
It takes a deep blue stain by "Wright's method. The proto- 
plasm is faintly granular and takes a blue stain. The transi- 
tional cell is believed by some to be identical with the large 
mononuclear but is usually distinguishable from the latter by 
the fact that its protoplasm is granular while that of the large 
mononuclear is non-granular. 

In the course of differential counting certain cells are oc- 
casionally met. with which are so distorted by the process of 
preparing the slides and the staining that they are unrecog- 
nizable. In such cases these distorted cells are simply classi- 
fied under a separate column headed "unclassified." 

Occasionally, in certain diseases (myeloid leukemia) or in 
any condition associated with a sudden outpouring of poly- 
morphonuclear leucocytes into the blood stream, certain young 
forms of these polymorphonuclear cells are noted in the blood. 
These forms are recognized by the fact that while they have 
the typical granular protoplasm characteristic of the neutro- 
phils, basophiles and eosinophils, and that the size and 
general appearance is similar, the neucleus is different since, 
instead of being polymorphous it is round or oval. Such cells 
are called ' ' myelocytes, ' ' and are of three varieties depending 
upon the character of their protoplasm : Neutrophilic, eosino- 
philic, and basophilic. 

VI. Widal Reaction. — 

Necessary Apparatus. — 

White mixing pipette. (Same as used for white blood count.) 

Blood sticker. 

Centrifuge tube. 

Centrifuge. 

Hollow grQund slides. (Hanging drop slides.) 

Cover-glasses for slides. 

Platinum loop. 



46 Clinical and Laboratory Technic 

Necessary Reagents. — 

12-hour growth of bacillus typhosus in bouillon. 
Physiological salt solution (0.8%). 

(1) Draw up blood to the mark "1" in the white count- 
ing pipette. Fill pipette quickly to the mark 11 with physio- 
logical saline solution. Shake. (2) Express mixture into a 
clean centrifuge tube and centrifuge for % to 1 minute. (3) 
Place one platinum loopful of the clear supernatant fluid upon 
a clean cover-glass. (4) Mix with this one platinum loopful 
of the 12-hour culture of bacillus typhosus. (5) Make a 
hanging drop of this on the hollow ground slide, being sure 
that the edges are air-tight. (Oil immersion or vaseline 
around edges.) (6) In a similar manner make a hanging 
drop of a loopful of bacillus typhosus culture and physiologi- 
cal saline solution. This is used as a control. (7) Examine 
both hanging drops under the microscope for clumping and 
loss of motility, at intervals of five minutes, for the next half 
hour. The reaction is considered positive if marked clump- 
ing associated with loss of motility occurs within this time 
and if the control drop remains unchanged. 

VII. Blood Cultures.— 

Necessary Apparatus. — ■ 

5 c.c. syringe with sharp needle. 

Tourniquet. 

Incubator. 

Necessary Reagents. — 

Two sterile flasks containing 200 c.c. each of bouillon. 

For the diagnosis of typhoid by blood culture, draw off 5 
c.c. of blood from the vein and place 2y 2 c.c. into each of two 
flasks containing 200 c.c. of bouillon. Incubate for 24 hours 
and examine a hanging drop at the end of this time for motile 
bacilli. If none are found re-incubate for 24 hours more and 
re-examine. If motile bacilli are present in both flasks a pre- 



Examination of the Sputum 47 

sumptive diagnosis of typhoid fever may be made. The evi- 
dence must always be confirmed, however, by inoculations into 
fresh bouillon and into glucose agar or glucose bouillon. (An 
agglutination test is done with the inoculated bouillon growth, 
after twelve hours' incubation, using a known typhoid serum. 
Gas formation is looked for in the glucose agar or glucose 
bouillon. If gas is present either the organism is not typhoid 
or there is a mixed infection present. If clumping occurs 
with a known positive serum in a dilution of 1-100, and if no 
gas is formed in glucose agar or bouillon, a diagnosis of ty- 
phoid fever may be made.) 

EXAMINATION OF THE SPUTUM. 

I. Gross Examination. — 

1. Color.— Bloody? Rusty? Green? Yellow? 

2. Odor. 

3. Consistency. — Mucous? Muco-purulent ? Purulent? 
Serous ? Foamy ? 

II. Microscopic Examination. — 
1. Unstained Sputum. — 

(a) Elastic Fibers. — (Occur in ulcerative tuberculosis, 
gangrene and abscess of the lung.) 

Pick out any whitish or grayish masses which may look 
promising. Press between cover-glass and slide and examine 
with low power for the shining, refractile, branching elastic 
fibers. 

If it is desired to make a careful search for elastic fibers, 
mix equal parts of sputum and 10% sodium hydroxide in a 
test tube. Warm slightly, but do not boil, until fairly clear. 
Pour into centrifuge tube, centrifuge and examine sediment 
for branching, refractile elastic tissue fibers. 

(b) Other Abnormalities. — Curschmann's spirals? Para- 
sites? Moulds? 



48 Clinical and Laboratory Technic 

-2. Tubercle Bacillus. — 

There are numerous methods of staining for the tubercle 
bacillus, all of which are more or less satisfactory. "We give 
only two of them, for the sake of brevity, both of which are 
good and are in general use. 

(a) Pappenheim 7 s Method. — 

Necessary Apparatus. — 

Clean slide. 
Platinum wire. 
Cover-glass forceps. 

Necessary Reagents. — 

Carbol-fuchsin.* 
Pappenheim 's solution.* 
*For directions as to making stains, see appendix. 

(1) Make a thin smear of suspected sputum, being care 
ful to pick out any grayish or whitish masses present. Dry. 
Fix by heat. 

(2) Cover with carbol-fuchsin and boil for one minute or 
place in a dish of carbol-fuchsin ( Czaplewsky 's carbol-fuchsin) 
for 15 minutes. 

(3) Wash in water. 

(4) Apply Pappenheim 's stain for two minutes. 

(5) Wash in water. 

(6) Apply Pappenheim 's stain again for iy 2 minutes. 

(7) Wash in water, blot until dry, and examine under oil 
immersion for red, acid-fast bacilli. 

(b) Acid Alcohol Method. — 
Necessary Reagents. — 

Carbol-fuchsin.* 

Acid alcohol. (2% hydrochloric acid in 80% alcohol.) 
*For directions as to making stains, see appendix. 



Examination of the Sputum 49 

(1) Prepare smear as in preceding method. 

(2) Stain with carbol fuchsin as in preceding method. 

(3) "Wash in water. 

(4) Apply acid alcohol until all of the smear is decolor- 
ized except the thickest parts. 

(5) Wash in water. 

(6) Apply Loeffler's methylene blue* for two minutes. 

(7) Wash in water. 

(8) Dry and examine under oil immersion for character- 
istic acid-fast (red) bacilli. 

(e) Antiformin Method of Concentrating Tubercle Bacilli 
in Sputum. — 

Xccessary Apparatus. — 

Centrifuge (preferably electric). 
Clean centrifuge tube. 

Xccessary Reagents. — 

Antiformin (a proprietary preparation marketed by the Amer- 
ican Antiformin Co., New York). 
Chloroform. 
Alcohol (95%). 

Equal parts of sputum and 40% antiformin are mixed in a 
test tube, about 2 c.c. of each being sufficient. This mixture 
is then boiled until all masses of sputum have been dissolved, 
adding, if necessary, additional antiformin solution in order 
to dissolve all sputum. As soon as this has been accomplished, 
cool the mixture by holding the tube under running water. 
Now add about 2 c.c. of a mixture of 10% chloroform in 95% 
alcohol; pour into a centrifuge tube and centrifuge at high 
speed for about ten minutes (1500 R. P. M. or over). 

After centrifugalization three layers will be formed in the 
centrifuge tube. The top layer is poured off and the second 



*For directions as to making stains, see appendix. 



50 Clinical and Laboratory Technic 

layer which will consist of a disk floating upon the clear 
bottom layer of chloroform, is expressed upon a clean slide. 
This layer will contain any tubercle bacilli which may be 
present. 

Since all mucin has been dissolved by the antiformin, it is 
necessary to use some fixing medium with this preparation be- 
fore it can be stained. This is best accomplished by simply 
adding a small amount of the untreated sputum to the slide, 
mixing well with the material to be examined, fixing by heat 
and staining in the usual way. 

3. Influenza Bacillus. — 

Necessary Reagents. — 

Carbol-gentian violet.* 
LugoPs solution.* (Gram's solution.) 
95% alcohol. 

2% safranin or dilute carbol-fuchsin (1-10).* 
*For directions as to making stains, see appendix. 

(a) Make a thin smear of suspected material, dry and fix 
by heat. 

(b) Apply carbol-gentian violet for two minutes. 

(c) Apply Lugol's solution for one minute (without wash- 
ing). 

(d) Apply 95% alcohol until blue color ceases to come 
out. (Usually about 30 seconds is sufficient, the time depend- 
ing, however, upon the thickness of the smear.) 

(e) Wash in water. 

(f) Counterstain with 2% safranin or with dilute carbol- 
fuchsin (1-10) for one or two minutes. 

Influenza bacilli appear as short, gram-negative (reddish) 
bacilli. They are always found in groups and are often intra- 
cellular. Unless large numbers of such bacilli are present in 
the smear they are not considered of great diagnostic sig- 
nificance. In order to differentiate the influenza bacillus with 
absolute certainty a culture should be made from the sputum. 
As a rule, however, this is not done, the smear alone being re- 
lied upon. 



Examination of the Sputum 51 

4. Pneumococcus. — 

Stain by Gram's method as for influenza bacilli. Pneumo- 
cocci appear as lanceolate-shaped, gram-positive diplococci, 
(staining bine) . Unless large numbers of these cocci are found 
in the smear, no diagnostic significance should be attached to 
their presence since they are found in many normal mouths 
in small numbers. 

5. Diphtheria Bacillus. — 
Necessary Reagents. — 

Loeffler's methylene blue.* 
*For directions as to making stains, see appendix. 

(a) Make smear from throat, taking material from under 
the edge of membrane if there is a membrane present. 

(b) Dry and fix by heat. 

(c) Cover with Loeffler's methylene blue for two minutes, 
warming slightly, but not boiling. 

(d) Wash with water, dry and examine under oil immer- 
sion for characteristic barred, beaded or clubbed bacilli. 

6. Diphtheria Cultures. — 

No absolute diagnosis of diphtheria can be made from smears 
taken directly from the throat. Although the presence of 
large numbers of "diphtheroids" in such a smear is sufficient 
for a presumptive diagnosis and, in a suspicious case, would 
justify the administration of antitoxin, a negative report af- 
ter such an examination would be of absolutely no value. At 
the time the smear is made from the throat, therefore, a cul- 
ture should also be made. 

Necessary Apparatus. — 

Incubator. 

A tube of Loeffler's blood serum media. 

The smear is taken from under the edge of the membrane 
and is rubbed over the surface of the culture media. The 



52 Clinical and Laboratory Technic 

tube is then incubated for from eight to twelve hours at the 
end of which time any suspicious colonies are transferred to 
a slide and stained as directed for diphtheria bacilli. If the 
culture is positive numerous "diphtheroids" will be seen. A 
culture which has been incubated for much more than twelve 
hours after it was taken from the throat is rarely of great 
diagnostic value, even though diphtheria bacilli were pres- 
ent, since, after that length of time, the other organisms pres- 
ent in the throat begin to overgroAV the diphtheria bacilli, of- 
ten obscuring them entirely. 

EXAMINATION OF THE GASTRIC CONTENTS. 

I. Empty Stomach Contents. — 

The contents of the empty stomach are best taken in the 
early morning before the patient has taken anything whatever 
by mouth. The best and easiest method of obtaining contents 
from the empty stomach is by means of one of the various 
modifications of the Einhorn duodenal tube. Normally, from 
10to50c.c. of a whitish, slightly turbid fluid may be obtained 
from the fasting stomach. The presence of food particles af- 
ter a fast of from eight to twelve hours means obstruction to 
the pylorus as a rule. (In order to obtain a satisfactory micro- 
scopic picture of the cellular elements in the gastric contents 
this method must be used.) 

1. Free Hydrochloric Acid. — (In the normal empty stom- 
ach the percentage of free HC1 varies from to 20.) 

Necessary Apparatus. — 

Burette and stand. 

White porcelain dish. 

10 c.c. graduated cylinder. 

Necessary Reagents. — 

Tenth normal sodium hydroxide. 
Topfer's reagent. (Dimethyl-amido-azobenzol .5% 
alcoholic solution.) 



Examination of the Gastric Contents 53 

Place exactly 10 c.c. of the unfiltered gastric contents in a 
porcelain dish. Add two or three drops of Topfer's reagent. 
If free hydrochloric acid is present a distinct red color will be 
produced. If a red color is formed on the addition of Top- 
fer's reagent, add tenth normal sodium hydroxide from the 
burette until this red color has entirely disappeared, its place 
being taken by a light canary yellow. The complete disap- 
pearance of all traces of red marks the end of the reaction 
and means that all free hydrochloric acid has been neutralized 
by the sodium hydroxide. The amount of sodium hydroxide 
which was used in neutralizing this free hydrochloric acid is 
carefully noted in cubic centimeters and tenths of a cubic 
centimeter. The acidity is then expressed, for convenience, 
by multiplying this figure by ten. For example, if it re- 
quired 2.5 c.c. of tenth normal sodium hydroxide to neutralize 
the free hydrochloric acid in a given specimen, the free hydro- 
chloric acid percentage would be 2.5 multiplied by 10 or 25. 

2. Total Acidity. — 

Necessary Apparatus. — 

Same as for free HC1. 

Necessary Reagents. — 

1% phenolphthalein solution. (Alcoholic solution.) 

The total acidity of the gastric contents is obtained from 
the same preparation which was used in the determination 
of the free hydrochloric percentage. 

As soon as the red color, caused by the free hydrochloric 
acid, has disappeared from the mixture and the exact amount 
of sodium hydroxide which was used in bringing about this 
disappearance has been noted, a few drops of phenolphthalein 
solution are added, and the addition of tenth normal sodium 
hydroxide is continued until a permanent red is obtained (be- 
ing careful to stir while the sodium hydroxide is being added). 
To determine the total acidity, now, we simply add the amount 
of tenth normal sodium hydroxide used in neutralizing the 



54 Clinical and Laboratory Technic 

free hydrochloric acid to the amount used afterwards in bring- 
ing about the permanent red color- with the phenolphthalein 
solution. For example: If in a given case, 2.5 c.c. of tenth 
normal sodium hydroxide were used in neutralizing the free 
hydrochloric acid and one and two-tenths c.c. (1.2 c.c.) were 
required, in addition, before the solution took on a red color 
with phenolphthalein, the total acidity would be, 2.5 plus 1.2 
which is 3.7, and which is expressed as "37." 

3. Lactic Acid. — 

Kelling's Test. — 
Necessary Reagents. — 

Watery solution of ferric chloride. 
Distilled water. 

To a test tube full of distilled water, add just enough of a 
solution of ferric chloride to give it a faint yellow color. (One 
or two drops are sufficient as a rule.) Pour one-half of this 
solution- into another test tube. To one of these test tubes 
add a small amount of the filtered gastric contents which are 
to be tested. Use the other tube as a control. If lactic acid 
is present a distinct yellowish color develops as soon as the 
gastric contents are added, while the control tube remains, of 
course, unchanged. 

4. Occult Blood. — 

(a) Guaiac Test. — ■ 
Necessary Reagents.— 

Glacial acetic acid. 
Fresh tincture of guaiac* 
Fresh hydrogen peroxide. 

*Made by adding a small particle of gum guaiac to a small amount (1 to 2 c.c.) 
of 95% alcohol and shaking well. 

(1) To several cubic centimeters of gastric contents (1 to 
3 c.c), add x /2 c.c. of glacial acetic acid. Shake. Add a small 
amount of ether (1 to 2 c.c). Shake. 



Examination of the Gastric Contents 55 

(2) To 2 c.c. of fresh tincture of guaiac add fresh hydro- 
gen peroxide until a white milky color is obtained. 

(3) Pour the ethereal extract of the gastric contents into 
the guaiac-peroxide solution. In the presence of blood, a deep 
green color forms at once. 

(b) Benzidine Test.— 
Necessary Reagents. — 

Fresh tincture benzidine.* 

Fresh hydrogen peroxide. 

Glacial acetic acid. 

*Add a small quantity of powdered benzidine to a few e.c. of 95% alcohol and 
shake well. 

Mix equal parts (1 or 2 c.c. each) of (1) fresh tincture ben- 
zidine, (2) fresh hydrogen peroxide and (3) pure gastric con- 
tents. Shake well and add a few drops of glacial acetic acid. 
If blood is present, a deep, greenish blue will make its appear- 
ance promptly. 

(c) Meyer's Test.— 
Necessary Reagents. — 

Phenolphtlialein 4 gms. 

Potass, hydroxide 35 gms. 

Distilled water 200 c.c. 

Mix and allow to ' stand 3 minutes. Then add pure zinc 
dust, 20 gms., and boil until decolorized. Keep in a dark, 
well stoppered bottle, along with the zinc dust. 

Place equal parts of this solution and the solution to be 
tested in a test tube. Shake and add a few drops of hydro- 
gen peroxide. If test is positive for blood, a pink color will 
form upon the addition of the hydrogen peroxide. 

II. Microscopical Examination of the Gastric Contents. — 

It is necessary, in order to obtain a satisfactory picture of 
the cellular elements of the gastric contents, to examine only 
the contents of the perfectly empty stomach microscopically. 



56 Clinical and Laboratory Tecknic 

since food, water, residue of test meals, etc., will entirely ob- 
scure the microscopic picture. Moreover, it is necessary to 
make the microscopic examination directly the contents are 
removed from the stomach, since the important cellular ele- 
ments are very rapidly digested in the presence of hydro- 
chloric acid and pepsin. 

1. Examination of the Fresh Contents. — 

(a) Increase of mucous? (A few strands of mucous, con- 
taining a few enmeshed leucocytes, are normal.) Thick, ropy 
contents of the empty stomach are pathological. 

(b) Increase of pus cells? 

(c) Eed blood cells? 

(d) Gastric epithelial cells? 

(e) Yeasts? Sarcinse? Bacteria? 

(f) Food particles? 

2. Stained Gastric Contents. — 

Contents after a test meal or empty contents may be used. 
Stain by Gram's method. (For directions see under Sputum.) 

(a) Oppler-Boas bacilli. (Thick, Gram positive bacilli, 
occurring in chains.) 

(b) Yeasts and sarcinae. 

III. Gastric Contents After Test Meals. — 

If a test meal is given the contents should be removed from 
one-half to one hour after giving the meal. The test meal in 
most common use is the "Ewald, " consisting of a large slice 
of bread and a glass and a half of water. All of the chem- 
ical tests described above should be carried out on the gas- 
tric contents removed after a test meal. It is well to bear in 
mind, however, in testing the contents of the stomach for 
lactic acid after a test meal, that the test meal itself is apt 
to contain traces of lactic acid, so that no conclusions should 
be drawn as to its significance until the contents of the empty 
stomach are tested. 



Examination of tlie Duodenal Contents 57 

EXAMINATION OF THE DUODENAL CONTENTS. 

Duodenal contents are obtained by means of the duodenal 
catheter. They are recognized by (a) an alkaline, neutral, or 
very slightly acid reaction to litmus paper; (b) a golden yel- 
low color due to bile and (c) the presence of pancreatic fer- 
ments. 

I. Gross Appearance. — 

1. Mucus. The contents should be fluid. Thick tenacious 
mucous contents usually mean a pathological condition (du- 
odenitis). 

2. Turbidity. (Cholecystitis?) 

3. Blood. 

II. Microscopic Examination. — 

For a satisfactory microscopic examination, the duodenal 
contents must be examined immediately after removal as only 
a very few minutes are sufficient to allow the important cellu- 
lar elements to be so distorted by pancreatic digestion as to 
be unrecognizable. The reaction must, moreover, be alkaline 
or neutral to litmus since acid, by precipitating the bile salts, 
entirely destroys the microscopic picture. 

1. Bile-stained pus or epithelial cells. 

2. Bile-stained casts. 

3. Red blood cells. 

III. Chemical Examination. — 

1. Diastase (Starch splitting ferment). — 
Necessary Reagents. — 

Kahlbaum's soluble starch. 
Distilled water. 
Tincture iodine. 



58 Clinical and Laboratory Technic 

Dilute one c.c. of duodenal contents with 9 c.c. of distilled 
water, being sure that contents are either neutral or alkaline 
to litmus. 

Into each of six test tubes place 5 c.c. of a 1% starch solu- 
tion made by boiling 0.5 gm. of Kahlbaum's soluble starch in 
50 c.c. of distilled water. (This solution must be fresh.) To 
the first tube add 1 c.c. of the solution of duodenal contents, 
(10%) ; to the second .5 c.c; to the third .25 c.c. ; to the fourth 
1 c.c. and to the fifth .05 c.c. The sixth tube is used as a 
control. Incubate in the water bath for three-quarters of 
an hour, or in the incubator for iy 2 hours at 37 degrees Centi- 
grade. At the end of this time fill each tube with cool tap 
water and add to each a few drops of tincture of iodine. If 
diastase is present in normal quantities all tubes except the 
control will give colors ranging from yellow to purple due 
to the conversion of the starch to achro or erythro-dextrin. 
(Since an acid reaction will destroy diastase it is important 
to be certain that the duodenal contents which were used were 
not acid in reaction. If they were acid it is useless to test 
for diastase. With this exception, the absence of diastase is 
strong evidence of the presence of pancreatic disease.) 

2. Trypsin (Proteid splitting ferment). — 
Necessary Reagents. — 

Desiccated casein. 
5% acetic acid. 
Sodium bicarbonate. 

To each of six test tubes add 10 c.c. of a 1-1000 solution of 
dried casein (made by boiling .1 gm. of casein plus .1 gm. of 
sodium bicarbonate in 100 c.c. of distilled water for one min- 
ute). 

To the first of these tubes add 1 c.c. of a 10% solution of 
duodenal contents; to the second add .5 c.c; to the third add 
.25 c.c; to the fourth add .1 c.c. and to the fifth .05 c.c 
The sixth tube is used as a control. Incubate for three-quar- 
ters of an hour in the water bath or for iy 2 hours in the in- 
cubator. 



Examination of tlie Duodenal Contents 59 

At the end of this time add a few drops of dilute acetic 
acid (5%) to each tube. If trypsin is present in normal quan- 
tities no precipitate will form in any tube except the control 
on the addition of the acid, while if trypsin is absent all tubes 
will show a milky precipitate on the addition of acid. The 
absence of trypsin, even if the duodenal contents were acid in 
reaction, is excellent evidence of the existence of pancreatic 
trouble. 

3. Lipase (Fat splitting ferment). — 
Necessary Reagents. — 

Pure olive oil. 
Phenolphthalein solution. 
Tenth normal sodium hydroxide. 

Into each of two test tubes place exactly 10 c.c. of purest 
olive oil. 

To one of these tubes add 2 c.c. of pure duodenal contents. 
Use the other tube as a control. 

Incubate for three-quarters of an hour in the water bath 
or for iy 2 hours in the incubator. 

Add a few drops of phenolphthalein solution to each of the 
tubes at the end of this time, and titrate the acidity of the two 
with tenth normal sodium hydroxide. If lipase is present 
in the duodenal contents there will be a much higher acidity 
in the test tube than in the control tube, due to the setting 
free of the fatty acids (the olive oil being split into fatty 
acids and glycerine by lipase). This difference in the acidity 
of the two tubes is a rough measure of the amount of lipase 
present in the duodenal contents. Normally the difference 
varies from 150 to 500 (15 to 50 c.c. of tenth normal sodium 
hydroxide). Since an acid reaction of the duodenal contents 
will also destroy lipase, it is important, before interpreting a 
reaction, to be sure that the reaction of the duodenal contents 
was neutral or alkaline. 

It is better in doing the above tests to run all of them simul- 
taneously using 14 test tubes in all. The absence of any fer- 



60 Clinical and Laboratory Teclinic 

ment or even a marked diminution is' strong presumptive evi- 
dence of pancreatic mischief, granting (a) that the duodenal 
contents were fresh and (b) that they were not acid in reac- 
tion. If the contents are acid the absence of trypsin only is 
of value. 

EXAMINATION OF THE FECES. 
I. Appearance. — 

1. Color.— White? Black? Fresh blood? Yellow? 



2 



Consistency.— Formed ? Soft? Fluid? Foamy? Mu- 



cous? Fatty? 

II. Occult Blood.— 

1. Guaiac Test. — (See Gastric Contents.) 

2. Benzidine Test. — (See Gastric Contents.) 

3. Meyer's Test. — (See Gastric Contents.) 

III. Bile Pigments. — 

Schmidt 's Test. — 
Necessary Reagents. — 

Saturated aqueous solution of bichloride of mercury. 



Necessary Apparatus. 

Covered dish. 
Pestle and mortar. 



Cover 2 or 3 c.c. of the fresh stool with a saturated solution 
of bichloride of mercury. Grind up thoroughly with a pestle. 
Cover and alloAv to stand twenty-four hours. If bilirubin is 
present food particles will, at the end of twenty-four hours, be 
colored green. If hydro-bilirubin (urobilin) is present they 
will be stained red. (Examine macroscopically and micro- 
scopically, paying particular attention to color of mucous, epi- 
thelial cells, pus cells, etc.) 



Examination of tJie Feces 61 

IV. Ferments. — 
1. Diastase. — 

In suspected disease of the pancreas, especially in those 
cases where it is impossible to obtain satisfactory specimens 
of the duodenal contents, it is often possible to obtain diag- 
nostic information from an examination of the stools for the 
pancreatic ferments, especially for diastase. In order to ob- 
tain this information, however, it is necessary to place the 
patient upon a standard preliminary diet and to give a stand- 
ard "pancreatic meal." The preliminary treatment recom- 
mended by Brown has given us the best results.* It is given 
as follows: 

The patient must be upon a liquid diet for two days pre- 
ceding the test. On the night before the test give a high soap 
suds enema. 

At 7 a. m. give 750 c.c. (21 ounces) of whole milk. 

At 7 :30 a. m. give one-half ounce of Epsom salts. 

At 8 a. m. give one-half ounce of Epsom salts. 

At 8:30 a. m. give one-half glass of water containing one 
dram of sodium bicarbonate. 

At 1 p. m. give a high enema consisting of 500 c.c. of normal 
saline (one pint). If there is no result from this in three- 
quarters of aii hour give another of one quart of normal salt 
solution. 

Preserve all stools passed from 7 a. m. to 2 p. m. with the 
results of the enemata and send to the laboratory. On arriv- 
ing at the laboratory the stool is diluted up to 3,000 c.c. with 
normal saline, and a small portion is well centrifuged. The 
supernatant fluid is used in carrying out the tests, which is 
done as follows: 

Into each of six test tubes is placed 2 c.c. of 1% starch solu- 
tion (Kahlbaum's soluble starch). To the first tube add 1 
c.c. of the supernatant fluid ; to the second .5 c.c. ; to the third 
.25 c.c. ; to the fourth .1 c.c. and to the fifth .05 c.c. Use 



*Bro\vn: Johns Hopkins Hosp. Bull., 1914, xxv, 200. 



62 



Clinical and Laboratory Technic 



the sixth tube as a control. Incubate for 45 minutes in the 
water bath or for iy 2 hours in the incubator. At the end of 
this time fill all tubes with tap water and add a few drops 
(two) of tincture of iodine to each tube. If diastase is pres- 
ent in normal quantities all test tubes except the control will 
give a color change ranging from yellow to purple, while the 




Fig. 16. — Microscopic elements of normal feces, a, Muscle-fibers; b, Connective 
tissue; c, epithelial cells; d, White blood corpuscles; e, Spiral vessels of plants; 
f-h, Vegetable cells; i, Plant hairs; k, Triple phosphate crystals; I, Stone cells. 
Scattered among these elements are micro-organisms and debris (after v. Jaksch). 

control will be a deep blue. The persistent absence or marked 
diminution of diastase in the stool obtained in this manner is 
strong evidence of pancreatic trouble. 

2. Trypsin. — 

Trypsin may be tested for in the same manner as described 
under duodenal contents, using the supernatant fluid as in the 
test for diastase. 

3. Lipase. — 

Lipase is never present in the stools even in normal indi- 
viduals in our experience. 



V. Microscopic Examination. — 

The stool is always examined fresh and unstained. Mix a 



Examination of tJie Feces 



63 





Fig. 17. — Ova of ascaris lumbricoides, with and without envelope. 






Fig. 18. — Ovum of unci- Fig. 19. — Ovum of tri- Fig. 20. — Ovum of oxyuris 
naria americana. chocephalus dispar. vermicularis. 






Fig. 21. — Ovum of taenia 
saginata. 



Fig. 22. — Fntamceba histolytica. 



Fig. 23. — Cercomonas 
hominis. 




Fig. 24. — Ovum of 
taenia nana. 




Fig. 25. — Trichomonas. 



64 Clinical and Laboratory Technic 

small particle of stool (about the size of a match head) with 
three or four drops of water. Make a thin smear with a glass 
rod or toothpick, pour off the excess of material, and examine 
directly, while wet, first with the low power of the microscope, 
then with the high power for details of cells, ova, parasites, 
etc.* 

1. Pus cells? 

2. Red blood cells? 

3. Mucus? 

4. Azotorrhea (undigested muscle fibers) ? 

5. Abnormal amounts of fats? 

6. Parasites or ova? Do not give a negative report from 
the examination of one slide only, but search carefully through 
at least ten slides before rendering such a report. In looking 
for amebse, examine stool as soon as passed, or, if this cannot 
be done, pass a rectal tube, allow it to be retained for a few 
minutes and examine immediately any flecks of mucus brought 
down by the tube. 

VI. Extraction of Ova and Parasites from Stools. — 

Necessary Apparatus. — 

Centrifuge tube. ' 
Centrifuge. 
Small funnel. 
Gauze. 

Necessary Reagents. — 

25% antiformin.* 

Ether. 

*For directions as to making stains see appendix. (A proprietary preparation 
marketed by the American Antiformin Co., New York.) 

1. Ether- Antiformin Method. — 

(a) If stool is solid, mix a small portion with water until 
a soft semifluid mass is obtained. 



*The mistake most frequently made here is in the attempt to examine prepara- 
tions which are too thick. One should be able to see small print through such a 
preparation. 



Examination of Ihc Spinal Fluid 65 

(b) To 2 or 3 e.c. of this mass, add 7 to 10 c.c. of 25% 
antiformin. ' Mix well. 

(c) "Warm for a few minutes, but do not boil. 

(d) Cool in running water, and add from 7 to 10 c.c. of 
ether. Mix well by shaking. 

(e) Filter through- one layer of gauze, into a centrifuge 
tube and centrifuge for one minute. 

(f ) Four layers are formed. Pour off three upper layers. 

(g) Draw up the sediment in bottom of tube with a bulb 
pipette ; place on a slide and examine for ova or larvae. 

EXAMINATION OF THE SPINAL FLUID. 

Spinal fluid should be examined as soon after removal as 
possible. In most spinal fluids a few cells will always be de- 
stroyed by autolysis if the fluid is allowed to stand for any 
great length of time. Since many pathological fluids only 
contain a slightly increased cell count, it is therefore impor- 
tant to make the cell count before any of these cells may have 
disappeared. A specimen kept on ice over night, however, 
may be examined the next morning, if an immediate examina- 
tion is impossible. 

If blood is present in the spinal fluid it is not necessary to 
attempt any of the following tests, except the Wassermann 
reaction and the stains for tubercle bacilli and other bacteria. 
A cell count and proteid reaction cannot be correctly inter- 
preted with tlie presence of red blood cells in the spinal fluid. 

I. Cell Count.— 

Necessary Apparatus. — 

Counting chamber with cover-glass (as for blood counting). 
Pipette. 

Necessary Reagents. — 
5% acetic acid. 



66 Clinical and Laboratory Teclinic 

Always stir fluid well before beginning an examination in 
order to insure a good distribution of the cells. 

1. Place a Few Dkops Upon a Clean Slide and examine 
with the low power of the microscope. If red blood cells are 
present it is not necessary to proceed with the cell count, a 
report of "Red blood cells present" being rendered. If in 
doubt as to whether the cells seen are red blood cells or leu- 
cocytes, add a few drops (two or three) of 5% acetic acid to 
the preparation on the slide. Red cells will immediately dis- 
appear while white cells will become more distinct than be- 
fore. If no cells are noted on examination of such a fresh 
specimen, or if not more than an average of one or two cells 
to a "low-power" field is made out, the spinal fluid may be 
reported as "practically acellular," or normal, without any 
further attempt at cell counting. 

,2. If the Fluid Contains a Cellular Increase, however, 
a further cell count is indicated. This is done by adding a 
few drops, (two or three) of 5% acetic acid to 1 c.c. of spinal 
fluid, placing a drop of this mixture in an ordinary counting 
chamber, and applying the cover-glass as in counting blood. 
When this is done count the number of cells contained over 
the area of one square millimeter. This number, multiplied 
by ten (the thickness of the drop counted) , represents the num- 
ber of cells contained in one cubic millimeter of spinal fluid. 
Any count of under ten cells per cubic millimeter is considered 
within normal limits, while any count over ten per cubic milli- 
meter is considered pathological. 

II. Differential Count. — 

Necessary Apparatus. — 

Centrifuge tube. 
Centrifuge. 
Clean slide. 
Platinum loop. 

Necessary Reagents. — 

Distilled water. 
Wright's stain. 



L 



Examination of the Spinal Fluid 67 

If there is a cellular increase in the spinal fluid it becomes 
necessary to determine what cellular elements are increased. 
Since it is important, from a diagnostic standpoint, to know 
whether the mononeuclear or the polymorphoneuclear cells 
are in the majority, particular attention should be paid to 
these two elements. 

(a) Centrifuge several cubic centimeters of spinal fluid. 

(b) Spread sediment upon a clean slide. 

(c) Dry in the air and cover with Wright's stain. 

(d) Stain as for blood. (See under blood.) 

(e) Make a differential of at least 25 cells, counting more, 
however, if possible, reporting only the relative percentage 
of the mononeuclears and polymorphoneuclear s. It is neither 
necessary nor advisable to attempt any further classification 
of cells than this in spinal fluid. 

III. Stains for Bacteria. — 

1. If Polymorphoneuclears are Present in any consid- 
erable number in the spinal fluid, bacteria should be searched 
for with especial care. The same preparation which was used 
in the differential count may be used for this examination 
also. If any bacteria are noted, stain another smear by Gram's 
method and examine carefully for meningococci (Gram-nega- 
tive, intracellular diplococci) ; for pneumococci (Gram-posi- 
tive, lanceolate diplococci); influenza bacilli ; streptococci; 
staphylococci, etc. 

2. Stain for the Tubercle Bacillus. — 

If tuberculous meningitis is suspected (clinical symptoms, 
high lymphocyte count in the spinal fluid, etc.), a special 
search for tubercle bacilli should be made. 

(a) Allow several cubic centimeters of the spinal fluid to 
stand over night (twelve to twenty-four hours), either on ice 
or at room temperature. 

(b) At the end of this time a film of fibrin will have 
formed in the fluid. Pour this film out on a clean slide, tak- 



68 Clinical and Laboratory Technic 

ing particular care not to allow it to become twisted into a 
coarse strand. (Upon the obtaining of this thin film of fibrin 
depends the success of the test.) 

(c) Dry and fix by heat. 

(d) Stain for the tubercle bacillus in the ordinary man- 
ner. (See under sputum.) 

IV. Chemical Tests of the Spinal Fluid. — 

1. Test for Globulin. (Butyric acid test of Noguchi.) — 

(a) To one part (0.2 c.c.) of spinal fluid, add five parts 
(1 c.c.) of 10% butyric acid solution. 

(b) Boil slightly. 

(c) Add one part (0.2 c.c.) of normal sodium hydroxide 
and reboil slightly. 

In the presence of an excess of globulin a flocculent pre- 
cipitate will form either immediately or within a half an 
hour. This test is positive in all forms of meningitis ; usually 
in spinal syphilis, tabes dorsalis and general paresis. 

2. Wassermann Reaction. — (Usually positive in tabes, 
general paresis and syphilitic meningitis.) 

3. Colloidal Gold Test for general paresis. (Lange's 
test.) 

4. Fehling's Test. — (See under urine for directions as 
to doing Fehling's test.) 

Normal spinal fluid should reduce Fehling's solution. The 
failure to reduce Fehling's solution occurs most often in the 
various forms of meningitis. The test is variable and is of 
little practical diagnostic value. 

EXAMINATION OF SEROUS EXUDATES AND 
TRANSUDATES. 

Any exudate or transudate (ascitic fluid, pleural fluid, peri- 
cardial fluid) should be examined immediately after removal, 
since clotting will often occur in a very few minutes, prevent- 
ing a satisfactory examination. 



Examination of Serous Exudates and Transudates 69 

I. Microscopic Examination. — 

1. Cell Count. — 

Place a few drops of the fluid upon a clean slide and ex- 
amine under the microscope for the presence of cells. (Use 
either high or low power.) If no cells are present, or if only 
a few large endothelial cells are noted, report the fluid as 
normal or accllular. 

2. Differential Count.- — 

If other cells are noted than the large endothelial cells 
make a differential count. 

Necessary Apparatus. — 

Centrifuge tube. 
Centrifuge. 
Platinum loop. 

Necessary Reagents. — 

Wright's stain. 

(a) Centrifuge a small amount of fluid (2-10 c.c). 

(b) Make a thin smear. 

v (c) Dry in the air and cover with Wright's stain. 

(d) Stain as for blood (see blood). 

(e) Make a differential of from 25 to 100 cells. (If red 
blood cells are present include them in the differential count.) 

3. Staining for Bacteria. — 

If there is a relative increase of the polymorphoneuclear 
cells look very carefully for bacteria during the cell count. 
If any bacteria are found, make another smear and stain by 
Gram, looking especially for pneumococci, streptococci, staphy- 
lococci, B. coli, etc. 

4. Staining for the Tubercle Bacillus. — 

If there is a relative increase in the percentage of mono- 
nuclear cells or of lymphocytes in the spinal fluid, one should 



70 Clinical and Laboratory Teclinic 

stain for the tubercle bacillus. This is done in the usual man- 
ner, drying a thin smear of sediment obtained by centrif uging 
several cubic centimeters, fixing by heat and staining as de- 
scribed under "sputum." 

The tubercle bacillus is rarely found in pleural or ascitic 
exudates, however, even when they are of known tuberculous 
origin. If it is desired to demonstrate them, therefore, in such 
exudates, it is best to inoculate a guinea-pig intraperitoneally 
with from 2 to 4 c.c. of the suspected fluid. If the exudate 
was tuberculous the guinea-pig will usually develop the dis- 
ease within six weeks, at the end of which time he should be 
autopsied. 

THE WASSERMANN REACTION. 
I. Various Modifications. — 

Two general types of Wassermann reactions are now ill 
common use. Both are valuable and reliable when properly 
carried out and it is probable that there is very little dif- 
ference as to their specificity for syphilis when the proper 
technic is used. The two general types are the old, or original 
Wassermann, in which sheep corpuscles are used as the basis 
of the reaction ; and the Noguchi modification of this reaction 
in which human red blood cells instead of sheep blood cells 
are used as the basis of the test. 

Since the Noguchi modification is the simpler of the two 
tests, since it is more easily carried out owing to the fact that 
human blood cells are always at hand while sheep blood is 
often difficult to obtain in the fresh state, and since, more- 
over, it would seem to be just as reliable as the original Was- 
sermann reaction it is perhaps in more general use in the 
South than is the original test. The original Wassermann, on 
the other hand, with its modifications, is probably in more 
general use in the larger Northern and Eastern medical cen- 
ters. 

For the sake of brevity we shall describe only that type of 
Wassermann reaction which is based upon the hemolysis of 



The Wassermann Reaction 71 

human red blood cells, which is the type of reaction used as 
a routine in this clinic. 

II. Apparatus. — 

The glassware used in this test should be both sterile and 
dry. While a drying oven is not absolutely necessary it is 
desirable. In the absence of such an oven the glassware may 
be cleaned by boiling in plain water, without soap or alkalis, 
then rinsing in distilled water. In any case they should al- 
ways be carefully rinsed first in tap water then in distilled 
water and then either dried in a drying oven or boiled in dis- 
tilled water. 

Necessary Apparatus. — 

lee box. 

Incubator or large water bath. 

Small Wassermann tubes. 

Racks for these tubes. 

At least V2 doz. 1 c.c. pipettes, graduated in y 100 c.c. 

At least 3 10 c.c. pipettes, graduated in y 10 c.c. 

Graduated centrifuge tubes. (15 c.c. capacity.) 

Graduated cylinder, 100 c.c. capacity. 

Petri dishes. 

Glass tubing, 4-6 mm. diameter (several lbs.). 

Centrifuge (preferably electric). 

Guinea pigs with pen for same. 

III. Reagents. — 

Necessary Reagents. — 

Known negative serum. 

Known positive serum. 

Antigen. (Acetone insoluble fraction of human heart extract.) 

Hemolytic amboceptor. (Rabbit serum which is hemolytic to 

human red blood cells.) 
Guinea pig serum (fresh). 
Washed red blood cells, human (fresh). 
Sterile normal saline solution (0.9%). 
Distilled water. 



72 Clinical and Laboratory Technic 

1. Known Negative Serum. — 

This may be obtained from any normal individual. Draw 
off several cubic centimeters of blood into a sterile syringe and 
express into a sterile bottle or test tube. Tip the receptacle 
over on its side before blood clots and allow to stand at room 
temperature for several hours, then place in the ice box over 
night. The next morning pipette off the clear serum which 
has separated from the clot, place in a sterile test tube or 
bottle or ampoule and inactivate by heating in a water bath 
for one-half hour at 56° C. This serum, if kept cool and 
sterile, may be used for weeks or months as a stock solution. 

2. Known Positive Serum. — 

Obtained and prepared in the same way as the known neg- 
ative serum except that it must be obtained from a patient 
who is known to have active syphilis, preferably from one in 
the secondary stage of the disease. This serum must also be 
inactivated in the water bath at 56° C. for one-half hour. 
(Unless one has access to plenty of patients, it is often diffi- 
cult to obtain satisfactory positive serum. It can be readily 
obtained, however, from any of the larger biological manu- 
facturing houses or from many of the larger Wassermann 
laboratories, at a fairly reasonable price.) 

3. Antigen. — 

For complete directions as to the making of the antigen 
used in these tests the reader must be referred to Noguchi's 
1 ' Serum Diagnosis of Syphilis. ' ' The manufacture of antigen 
requires some considerable time and trouble, so that it is prob- 
ably preferable, unless one is doing a large number of "Was- 
sermanns, to buy the antigen ready made. Antigen made 
from the acetone insoluble fraction of the human or beef 
heart may be obtained at a moderate outlay from any large 
biological manufacturing house or from any large commer- 
cial laboratory. The antigen comes dissolved in alcohol and 



The Wasser»i<nin Reaction 73 

ready for use. It is very stable if kept on ice and may be 
used for months as a stock solution. 

4. Hemolytic Amboceptor (Rabbit serum hemolytic to hu- 
man red blood cells). 

This is the most important and at the same time the most 
difficult of manufacture and preservation of any single re- 
agent used in the Wassermann test. Fortunately, however, 
it may also be obtained from many of the larger manufac- 
turers of biological products as well as from many of the 
larger commercial laboratories. It is prepared by injecting 
rabbits either intravenously or intraperitoneally with ascend- 
ing doses of washed human red blood cells. As a rule intra- 
peritoneal injections are preferred since the rabbits seem less 
likely to die from anaphylaxis after such injections. In im- 
munizing intraperitoneally one begins with an initial injec- 
tion of 5 c.c. of washed human corpuscles, in five days 8 c.c. 
are given, in five more days 12 c.c, in five more days 16 c.c. 
and finally after five more days 20 c.c. are given. The rabbit 
is then allowed to rest for from one week to ten days, when 
he is anesthetized and bled to death, the blood being collected 
in a sterile receptacle. It is then allowed to clot at room 
temperature, kept on ice for a few hours, and the serum 
pipetted off under asceptic precautions. Unless the serum 
is strongly hemolytic, it cannot be used in the Wassermann 
test and another rabbit must be immunized.* 

5. F resit Guinea-Pig Serum — Complement. — 

This must always be fresh. It is obtained by anesthetizing 
a full grown, healthy guinea-pig and bleeding him, either 
by cutting the carotid and bleeding to death or, preferably, 
by bleeding directly from the heart with a sterile syringe. 

*A very good hemolytic serum will hemolyze one c.c. of a 0.5% suspension of 
red blood cells, after one hour's incubation at 37° C. in a dilution of from one- 
tenth to one-fiftieth of a capillary drop, granted that the proper amount of com- 
plement has been added (one-tenth c.c. of a 40% solution of fresh guinea-pig 
serum). Hemolytic serum may be used in the Wassermann reaction, however, 
when it is much weaker than this. It should be discarded when it requires more 
than one-fourth of a capillary drop to hemolyze the given quantity of red blood 
cells in the given time (1 c.c. of a 0.5% suspension in one hour at 37° C). This 
serum when kept cool and sterile may be used for months as a stock solution. 



74 Clinical and Laboratory Technic 

The latter method has the advantage of not killing the pig 
if it is properly carried out. Four or five c.c. of blood may 
be very easily obtained in this manner in one or two minutes 
from an anesthetized pig without any apparent injury to the 
animal. Considerable practice is necessary before one becomes 
proficient in this technic, but after it is once acquired it saves 
much time and many guinea-pigs. As soon as the blood is 
obtained from the pig, regardless of the manner in which it 
was obtained, it is quickly expressed into a sterile petri dish 
and allowed to clot at room temperature. It is then placed 
on ice for from six to eighteen hours as convenient, at the 
end of which time the clear serum is pipetted off into a sterile 
tube when it is ready for use. This serum deteriorates in a 
very few hours even when kept on ice. It may be kept un- 
changed for days or weeks, however, if maintained in a frozen 
state. This may be very readily and economically done by 
filling an ordinary $1.00 thermos bottle with chipped ice mixed 
with table salt, inserting the test tube containing the guinea- 
pig serum into the mixture, corking well and placing the whole 
in the ice box. By replenishing the chipped ice and salt every 
third day the serum is kept constantly frozen and is ready 
for use at any time by simply allowing it to melt at room tem- 
perature, taking out the required amount and replacing the 
remainder in the chipped ice mixture. "We have kept serum 
over a period of six weeks in this manner without noticing 
any change whatever in its strength. 

For use in the Wassermann reaction the guinea-pig serum 
must be diluted one and a half times with normal saline solu- 
tion, making a 40% solution. 

6. Washed Red Blood Cells. — ■ 

These should be used fresh for each test. The necessary 
blood is very easily obtained by pricking the finger and squeez- 
ing a few drops into a graduated centrifuge tube filled with 
sterile normal salt solution. Ten drops of blood are usually 
sufficient for making 50 c.c. of a 0.5% suspension of red 
cells. When the requisite amount of blood has been obtained 
the solution is carefully mixed with a small pipette and thor- 



Tlie Wassermann Reaction 75 

oughly centrifuged. "When all blood cells are thrown to the 
bottom of the centrifuge tube the clear supernatant ♦fluid is 
poured off. the tube is refilled with normal saline, the con- 
tents well mixed and recentrifuged. Again, after thorough 
centrifugalization, the supernatant fluid is poured off, the 
tube refilled, the contents well mixed and thoroughly centri- 
fuged. When the cells are well thrown down after this third 
washing note carefully on the graduated scale of the centri- 
fuge tube the exact quantity of washed blood cells present, 
using one-tenth of a cubic centimeter as a unit. In order to 
make a dilution of 0.5% simply add 20 c.c. of normal saline 
solution for each one-tenth c.c. of washed blood cells. The 
resulting suspension is mixed well in a sterile graduated 
cylinder. The suspension is now ready for use. 

If kept sterile and cool the corpuscle suspension may be 
used, also, on the following day, but after standing over 36 
hours it is always advisable to make a fresh suspension before 
carrying out another test. 

When the above reagents are prepared, we are now T ready 
to carry out the test. 

IV. Technic— 

1. Setting Up the Tubes. — 

In carrying out the Wassermann reaction two sets of con- 
trol tubes must always be used regardless of the number of 
unknown sera which are to be tested. These are the known 
positive and the known negative controls. For each serum 
to be tested, also, there must be one special control, so that 
for each unknown serum we must set up two tubes and then, 
after the required number of tubes have been prepared for 
these unknown sera, two tubes are added for the known nega- 
tive serum and two for the known positive serum. After 
these tubes have been put in place in a test tube rack and 
each pair of tubes carefully numbered and the name of the 
patient whose serum is to be tested is carefully noted opposite 
each number on a slip of paper, exactly one c.c. of the 0.5% 



76 Clinical and Laboratory Technic 

suspension of red blood cells is added to each tube by means 
of a sterile graduated 10 c.c. pipette. 

2. Adding the Complement. — 

To each tube in the rack is now added exactly one-tenth 
c.c. of a 40% solution of fresh guinea-pig serum. This is 
done by means of the small graduated one c.c. pipettes. Ex- 
treme care must be taken to get exactly one-tenth c.c. of com- 
plement in each tube. If a slip is made and too much com- 
plement is added throw the contents of the tube out, refill 
with blood cell suspension and then add the correct amount 
of complement. Considerable practice is required before 
these small quantities of fluid can be accurately handled, but 
in order to get reliable results it is absolutely necessary that 
accurate measurements be made, particularly in dealing with 
complement. 

3. Adding tJie Sera to tJie Tubes. — 

To each pair of tubes is now added its corresponding sera, 
reference being made to the list of names which was made out 
before setting up the tubes and extreme care being taken to 
get the right serum in the right tube. Exactly one-tenth c.c. 
of serum* which has been previously inactivated by heating 
at 56° C. for one-half hour in the water bath is added to each 
pair of tubes. Before adding the next serum to the next 
pair of tubes either discard the pipette used on the previous 
serum or else rinse the pipette very carefully first in dis- 
tilled water and then in normal saline. Finally, into the 
proper tubes place exactly one-tenth c.c. of the known nega- 
tive serum and one-tenth c.c. of the known positive serum. 

4.. Addition of Antigen. — 

From the stock alcoholic solution of antigen take enough 
to make up the solution required for the tests, the amount of 

*The serum which is to be tested is obtained in the usual manner by inserting 
a needle into the arm vein and withdrawing from 1 to 5 c.c. of blood (preferably 
over 1 c.c). This is quickly expressed into a sterile test tube and allowed tc 
clot at room temperature. It is then kept on ice until the serum has separated 
from the clot, the serum is pipetted off and inactivated in the water bath at 56° 
for one-half hour. 



The Wassermann Reaction 



77 



which will depend upon the number of sera which are being 
tested. A ten per cent solution is made of the stock solu- 
tion in normal saline, forming a turbid opaque mixture. Ex- 
actly one-tenth c.c. of this mixture is now added to each of 
the front tubes only. By adding the syphilitic antigen to 
the front tubes only each rear tube becomes a control for its 
corresponding front tube. 

The tubes are now shaken w r ell and are ready for the pre- 
liminary incubation. When ready to incubate therefore 
they will appear somewhat as follows, supposing that we are 
testing only one unknown serum: 





Serum to be Tested. 


Negative Control 




Positive Control. 




0.5% suspension of 


.5% suspension R. 


B. 


.5% suspension R. B. 




R. B. Cs. 1 c.c. 


Cs. 1 c.c. 




Cs. 1 c.c. 


Rear 


40% solution of G. P. 


40% solution G. 


P. 


40%; solution G. P. 


tubes. 


serum 1/10 c.c. 


serum 1/10 c.c. 




serum 1/10 c.c. 




Unknown serum (in- 


Known negative 


se- 


Known positive se- 




activated) 1/10 c.c. 


rum 1/10 c.c. 




rum 1/10 c.c. 




0.5% suspension of R. 


.5% suspension R. 


B. 


.5% suspension R. B. 




B. Cs. 1 c.c. 


Cs. 1 c.c. 




Cs. 1 c.c. 




40% solution G. P. 


40% solution G. 


P. 


40% solution G. P. 


Front 
tubes. 


serum 1/10 c.c. 


serum 1/10 c.c. 




serum 1/10 c.c. 


, Unknown serum (in- 


Known negative 


se- 


Known positive se- 




activated) 1/10 c.c. 


rum 1/10 c.c. 




rum 1/10 c.c. 




10% solution of anti- 


10% solution of anti- 


10% solution of anti- 




gen 1/10 c.c. 


gen 1/10 c.c. 




gen 1/10 c.c. 



5. Incubation. — 

The tubes are incubated either in an incubator for one hour 
at 37° C. or in the water bath for one-half hour at the same 
temperature. It is during this preliminary incubation that, 
if the Wassermann be positive, the complement becomes fixed 
or bound by the syphilitic serum. No change is visible, how- 
ever, to the naked eye in the tube in which this reaction takes 
place, all tubes appearing exactly the same after incubation 
as they did before. 

This reaction which takes place when the complement be- 



78 Clinical and Laboratory Technic 

comes bound, although it is not visible to the naked eye, may 
be represented graphically somewhat as follows : 

Let [^> represent complement. 

Let ^> <C represent syphilitic amboceptor (antibody). 

Let <^\ represent syphilitic antigen. 

Since syphilitic antibodies in the presence of fresh comple- 
ment and of syphilitic antigen become fixed or bound, the 
following reaction takes place: 



This reaction can occur, however, only in those tubes con- 
taining syphilitic serum or antibodies. Moreover, it can oc- 
cur only in the front tubes since it will be remembered that 
no syphilitic antigen was added to the rear tubes. 

6. Titration of tlie Hemolytic Amboceptor. — 

Since the above described reaction, in which the binding 
of the complement by the syphilitic antibody occurs, is not 
visible to the eye it becomes necessary to determine whether 
it has taken place and it is in the determination of this point 
that the hemolytic amboceptor comes into play. It will be 
remembered that this serum is obtained by immunizing rab- 
bits against human red blood cells and that a good hemolytic 
serum will hemolyze many times its quantity of red blood 
cell suspension. Also, it will be recalled by those familiar 
with biological reactions that no amboceptor can work un- 
less it has some free or unbound complement to work with 
it or to ' ' activate ' ' it as it is called. We know therefore that 
if the complement in our tubes is bound by the syphilitic 
amboceptor there will be none to activate the hemolytic am- 
boceptor and that no hemolysis can take place in the tubes 
containing the syphilitic sera, while in all of the other tubes, 
since the complement is still free in them, hemolysis will take 



The Wassermann Reaction 79 

place promptly as soon as the proper amount of hemolytic 
serum is added. Hemolysis, therefore, proves that the com- 
plement is unbound and means that the serum which was 
contained in that tube did not contain syphilitic antibodies. 

Before adding this amboceptor to the tubes which we are 
testing, however, we must first determine how much of it 
to add. The determination of this question constitutes the 
key to the accuracy of the Wassermann reaction so that it is 
necessary to proceed at this point with the utmost care. If 
we use too much of the hemolytic serum certain positive Was- 
sermanns will become doubtful or negative reactions, while 
if we use too little of the hemolytic serum negative reactions 
will become doubtful.. A careful titration of the hemolytic 
serum must therefore be done for each Wassermann test which 
is carried out. The titration of one day cannot be made to 
serve for the next day but must be repeated on each separate 
occasion. 

The titration is carried out as follows: 

(a) Set up a series of ten sterile Wassermann tubes. 

(b) Add to each tube one c.c. of a 0.5% corpuscle sus- 
pension. 

(c) Add to each tube one-tenth c.c. of a 40% solution of 
fresh guinea-pig serum. 

(d) To the first tube add % capillary* drop of hemolytic 
amboceptor ; to #2, 14 drop ; to tube #3, % drop ; to tube 
#4, Vie drop ; to tube #5, % 2 drop ; to tube #6, % drop ; 
to tube #7, Y 6 drop; to tube #8, % 2 drop; to tube #9, % 4 
drop ; to tube #10, % 8 drop. 

(e) After the addition of these fractional drops to each 

*These fractional drops are made by making serial dilutions of the hemolytic 
serum and normal salt solution. For example, in order to get the fractional drops 
mentioned above we set up a series of ten tubes. To tube #1 of this series is 
added two capillary drops of the rabbit serum and to tube #6 is added one drop 
of serum. Two capillary drops of normal saline solution are now added to each 
of the ten tubes. The solution in tube #1 is mixed carefully by drawing it up 
into the capillary tube to which a rubber bulb has been attached and gently ex- 
pressing it. Two drops from tube #1 are now added to tube #2, the contents 
of which are then mixed and two drops from it transferred to tube #3 and so 
on down the line to tube #6, when the process is repeated as at tube #1. It is 
taken for granted that the reader knows how to make a capillary tube from an 
ordinary piece of glass tubing. 



80 Clinical and Laboratory Technic 

of the ten tubes containing the washed cells and the comple- 
ment, shake them well and incubate either in the incubator 
for one hour or in the water bath for one-half hour at 37° 
C, examining carefully every five minutes for hemolysis and 
shaking gently on each inspection. At the end of the period 
of incubation carry the tubes to a good light and determine 
which tubes are completely hemolyzed. The last tube in 
which complete hemolysis has occurred is noted and it is the 
strength of hemolytic serum used in that tube which is to 
be added to each of the Wassermann tubes. By complete 
hemolysis is meant the destruction of every red blood cell in 
the tube. If only a very faint turbidity occurs in a tube 
which is otherwise apparently hemolyzed use the next higher 
dilution as the standard. For instance, if the tube contain- 
ing one-twelfth of a drop of hemolytic serum is completely 
hemolyzed while that containing one-sixteenth of a drop al- 
though apparently hemolyzed, still contains a very few cells 
take the one-twelfth of a drop as the standard. 

7. Addition of tlie Hemolytic Amboceptor to the Wasser- 
mann Tubes. — 

After the incubation of the Wassermann tubes for one hour 
in the incubator or one-half hour in the water bath at 37 ° 
C, add the proper amount of amboceptor to each tube in the 
series (the amount to be added being determined as above 
described) ; being very careful not to skip any tube. When 
the proper amount of amboceptor has been added to each 
tube, shake them well and replace in the incubator. 

8. Interpreting tlie Tests. — 

After reincubation of the tubes examine them every ten 
minutes for the first half hour, shaking at each examination. 
When all of the rear tubes are completely hemolyzed; when 
both front and rear tubes of the negative control serum are 
completely hemolyzed, and when the rear tube of the posi- 
tive control is completely hemolyzed, remove the rack from 
the incubator or water bath and make a preliminary read- 



Gonococcas Fixation Test 81 

ing. As a rule this preliminary reading can be made within 
one-half hour or less; if necessary, however, incubate for as 
long as two hours. If they do not come down by then the 
reaction is a failure and the tests will have to be repeated 
after more careful titration of the amboceptor. In a satis- 
factory reaction a syphilitic serum will show an inhibition 
of hemolysis in the front tube, the rear tube being, of course, 
always hemolyzed. A negative serum will show complete 
hemolysis in both tubes. After this preliminary reading the 
tubes are shaken and placed in the icebox where they are al- 
lowed to remain for from two to twelve hours, at the con- 
venience of the worker. After this time the final readings 
may be made. 

In making the final reading tubes showing 75% or more 
inhibition of hemolysis, that is, tubes in which 75% or more 
of the red blood cells are still intact are reported as posi- 
tive. Tubes showing from 50 to 75% inhibition are reported 
as weak positive and tubes showing from 25 to 50% inhibition 
are reported as doubtful. Under 25% inhibition is consid- 
ered negative. No reaction should ever be interpreted as 
positive unless the rear tube is completely hemolyzed and 
unless both negative control tubes are completely hemolyzed. 

GONOCOCCUS FIXATION TEST. 

The serum test for gonorrhea is carried out in exactly the 
same manner as is the serum test for syphilis except that a 
known positive serum to gonococcus fixation must be obtained 
instead of a known syphilitic serum and an antigen made 
from mascerated gonococci must be used instead of a syph- 
ilitic antigen. Both of these reagents may be bought from 
the larger biological houses or from the larger commercial 
laboratories. This test is of value only in chronic long-stand- 
ing cases of gonorrhea and especially in gonorrheal arthritis. 



APPENDIX. 
COMMON STAINS USED IN THE LABORATORY.* 

1. Carbol-fuchsin. — 

Basic f uchsin 1 gm. 

5% carbolic acid solution 100 c.c. 

Mix and keep in a well corked bottle. This stain is apt 
to spoil if kept too long. 

2. Carbol-fuchsin. (Czaplewsky's Modification.) — 

This modification has the advantage over the ordinary car- 
bol-fuchsin that it does not deteriorate with age, but can be 
kept for years without injury. Moreover, it is not necessary 
to use heat with it in staining tubercle bacilli. It is made 
as follows : 

Basic f uchsin 1 gm. 

Carbolic acid (crystals) 5 gms. 

Glycerine 50 c.c. 

Dissolve ; then add distilled water .... 50 c.c. 

Better results are obtained in staining the tubercle bacillus 
by this stain if the stain is applied cold for 15 minutes, rather 
than heated, as in staining with the ordinary carbol-fuchsin. 

3. Carbol-gentian Violet. — 

This stain has the advantage over the analine gentian violet 
that it is permanent, not deteriorating with age, and being 
ready for use, in staining by Gram, at all times. It is made as 
follows : 

Saturated alcoholic solution of gen- 
tian violet 10 c.c. 

2^% solution of carbolic acid 90 c.c. 

*A11 of the following stains are easily made in the ordinary laboratory, from 
the dry stains which can be obtained from any dealer in laboratory apparatus and 
supplies (as Bausch and Lomb Optical Co., N. Y. ; Spencer Lens Co., N. Y. ; 
Arthur H. Thomas Co., Phila. ; Eimer and Amend, N. Y., etc.). If it is not de- 
sired to make these stains, they may also be bought already mixed at a slightly 
higher price from the same houses. 

82 



Common Stains Used in tihi Laboratory 83 

4. Loeffler's Methylene Blue. — 

Saturated alcoholic solution of meth- 
ylene blue . 30 c.c. 

Aqueous solution of potassium hy- 
droxide (1-10,01)0) 100 c.c. 

5. Lugol's Solution (Gram's Solution). — 

Iodine 1 gm. 

Potassium iodide 2 gms. 

Distilled water 300 c.c. 

6. Pappenheim's Stain. — 

Rosolic acid (corallin) 1 gm. 

Absolute alcohol 100 c.c. 

Saturate with methylene blue. 

Add 20 c.c. of glycerine. 

7. Safranin. — 

Saf ranin 2 gms. 

Distilled water q. s. ad 100 c.c. 

8. Wright's Stain.— 

I. 

Sodium bicarbonate 1 gm. 

Ehrlich 's medicinally pure methylene 

blue 2 gms. 

Distilled water 200 c.c. 

Steam in Arnold sterilizer 45 mins. 



II. 

Eosin (yellowish, water soluble) . . 1 gm. 
Distilled water 1000 c.c. 



As soon as the methylene blue mixture has cooled add it to 
the eosin mixture, little at a time, stirring constantly. When 
a drop of this mixture, placed on filter paper, no longer shoAvs 
an eosin color in the areola, cover the mixture and allow to 
stand over night. Next morning filter through one laver of 



84 Clinical and Laboratory Teclinic 

filter paper, dry the paper in air, cut in strips and place 
in a bottle with 500 c.c. of absolute methyl alcohol. (Merck's 
alcohol. ) 

When ready to use, dilute a small quantity of this mixture 
with equal parts of absolute methyl alcohol. This stain is 
permanent if the bottle is kept well corked. 



INDEX 



Abdomen, physical examination of, 

20 
Acetone in urine, 29 
Gunning's test for. 29 
Legal 's test for, 29 
Acidity, estimation of, in gastric 

contents, 53 
Albumin in mine, 23 

heat and acetic acid test for, 23 
heat and nitric acid test for, 23 
quantitative, 24 
Robert's test for, 23 
Appendix, stains used in labora- 
tory methods, 82 



Bence- Jones bodies in urine, 25 
Benedict's test for sugar in urine, 

26 
Benzidine test : 

for blood in urine, 32 

for occult blood in feces, 60 

for occult blood in gastric con- 
tents, 55 
Beta-oxybutyric acid in urine, 30 
Bile in urine, 31 

Rosenbaeh's test for, 31 
Bile pigments in feces, 60 

Schmidt's test for, 60 
Blood, 38 

cultures, 46 

differential counting, 43 

examination of, 38 

hemoglobin estimation, 41 

in urine, 32 

leucocytes, classification of,- 43 

red counting^ 40 

white counting, 38 

Widal reaction of, 45 

Rosenbaeh's test for, 31 
Blood in urine, 32 

benzidine test for, 32 

guaiac test for, 32 

Meyer's test for, 32 
Butyric acid test for globulin in 
spinal fluid, 68 



Carbol-fuchsin, 82 



Carbol-fuchsin (Czaplewsky 's mod 
ification), 82 

Carbol-gentian violet, 82 

Cardiovascular system, physical ex- 
amination of, 19 

Casts in urinary sediments, 36 

Cell count in ssrous exudates and 
transudates, 19 

Cell count in spinal fluid, 68 

Chemical examination of duodenal 
contents, 57 

Chemical examination of Bpinal 
fluid, 68 

Chest, physical examination of, IS 

Color, of urine, 22 

Crystals in urinary sediment, 36 

Cultures, blood, 46 
diphtheria, 51 

Curschmaim 's spirals in sputum, 47 

D 

Diacetic acid in urine, 30 

Gerhardt's test, 30 
Diastase in duodenal contents, 57 
Diastase in feces, 61 
Diazo reaction of urine, 30 
Differential cell count in serous ex- 
udates and transudates, 69 
Differential cell count in spinal 

fluid, 66 
Differential counting in blood, 43 
Diphtheria bacilli in sputum, 51 
Diphtheria cultures, 51 
Doremus method for quantitative es- 
timation of urea in urine, 33 
Duodenal contents, 57 

chemical examination of, 57 

diastase in, 57 

examination of, 57 

gross appearance of, 57 

lipase in, 59 

microscopic appearance of, 57 

trypsin in, 58 

E 

Elastic fibers in sputum, 47 

Empty stomach contents, examina- 
tion of, 52 

Ether-antiformin method of extrac- 
tion of ova and parasites 
from stools, 64 

Examination, physical, 16 

85 



86 



Index 



Extraction of ova and parasites 

from stools, 64 
Extremities, physical examination 

of, 21 

F 

Family history, 12 
Feces, 60 

appearance of, 60 

bile pigments in, 60 

examination of, 60 

extraction of ova and parasites 
from, 64 

microscopic examination of, 60 

occult blood of, 60 

pancreatic ferments in, 61 
Fehling's test for sugar in urine, 25 
Fehling's test in spinal fluid, 68 
Fermentation test for sugar in 

urine, 26 
Free hydrochloric acid estimation in 
gastric contents, 52 

G 

Gastric contents, 52 

after test meals, examination of, 

56 
empty stomach contents, examina- 
tion of, 52 
estimation of free hydrochloric 

acid in, 52 
estimation of total acidity in, 53 
examination of, 52 
lactic acid in, 54 
microscopic examination of, 55 
occult blood in, 54 
Oppler-Boas bacilli, 56 
sarcinse in, 56 
yeasts in, 56 
General appearance in physical ex- 
amination, 17 
General considerations in history 

taking, 11 
Genito -urinary system, physical ex- 
amination of, 20 
Gerhardt's test, for diacetic acid in 

urine, 30 
Globulin in spinal fluid, test for, 68 
Gonococci in urinary sediments, 37 
Gram's solution, 83 
Gross appearance of duodenal con- 
tents, 57 
Gross appearance of sputum, 47 
Guaiac test : 

for blood in urine, 32 

for occult blood in feces, 60 



Guaiac test — Con 't. 

for occult blood in' gastric con- 
tents, 54 
Gunning's test for acetone in urine, 
29 4 

H 

Head, physical examination of, 17 
Heat and acetic acid test for al- 
bumin in urine, 23 
Heat and nitric acid test, 23 
Hemoglobin estimation in blood, 41 
History taking, 11 

complaint, 12 

family history, 12 

general considerations, 11 

present illness, 14 

previous history, 13 



Indiean in urine, 31 

Obermayer's test for, 31 
Influenza bacilli in sputum, 50 
Influenza bacilli in spinal fluid, 67 

K 

Kelling's test for lactic acid in gas- 
tric contents, 54 



Laboratory methods: 

examination of blood, 38 

examination of duodenal contents, 
57 

examination of feces, 60 

examination of gastric contents, 
52 

examination of serous exudates 
and transudates, 69 

examination of spinal fluid, 65 

examination of sputum, 47 

examination of urine, 22 

gonococcus fixation, 81 

stains used in, 82 

Wassermann reaction, 70 
Lactic acid, in gastric contents, 54 

Kelling 's test for, 54 
Legal 's test for acetone in urine, 29 
Leucocytes in blood, classification 
of, 43 

large mononuclear, 44 

lymphocyte, 44 

polymorphonuclear basophile, 44 

polymorphonuclear eosinophile, 43 

polymorphonuclear neutrophile, 43 

transitional, 45 



Index 



ST 



Leucocytes in urinary sediments, 36 
Lipase in duodenal contents, 59 
Lipase in feces, 62 
Loeffler's methylene blue, 85 
Lugol's solution, 83 

M 

Meningococci in spinal fluid, 07 
Methylene blue. Loeffler's, 83 
Meyer 's test : 

for blood in urine, 32 

for occult blood in gastric con- 
tents, 55 

for occult blood in feces, 60 
Microscopic examination: 

of duodenal contents, 57 

of feces, 62 

of fresh gastric contents, 56 

of sediments in urine, 34 

of serous exudates and transu- 
dates, 69 

of stained gastric contents, 56 
Microscopic examination of sputum, j 
47 

Cursehmanu 's spirals, 47 

diphtheria bacilli, 51 

diphtheria cultures, 51 

influenza bacilli, 50 

moulds, 47 

parasites, 47 

pneumococci, 51 

stained, 48 

tubercle bacilli, 48 

unstained, 47 
Moulds, in sputum, 47 

N 

Neck, physical examination of, 17 
Nervous system, physical examina- 
tion of, 17 

. O 

Occult blood in feces, 60 

benzidine test for, 60 

guaiac test for, 60 

Meyer's test for, 60 
Occult blood in gastric contents, 54 

benzidine test for, 54 

guaiac test for, 55 

Meyer 's test for, 55 
Obermayer 's test for indican in 

urine, 31 
Oppler-Boas bacilli in gastric con- 
tents, 56 
Ova, extraction of, from stools, 64 I 



P 

Pancreatic ferments in feces, (il 
Parasites, extraction of, from stools, 
64 

Parasites in sputum, 1 i 

Pappenheim's stain, 83 
Physical examination, 16 

abdomen, 20 

cardiovascular system, 19 

chest, is 

extremities, 21 

general appearance, 17 

general remarks, 16 

genito-urinary system, 20 

head, 17 

neck, 17 

nervous system, 17 

rectum, 21 
Pneumococci in spinal fluid, 67 
Pneumococci in sputum, 51 
Polymorphonuclear basophile leu- 
cocytes in blood, 44 
Polymorphonuclear eosinophile leu- 
cocytes in blood, 43 
Polymorphonuclear neutrophil leu- 
cocytes in blood, 43 
Present iliness, 14 
Previous history, 13 

Q 

Quantitative albumin in urine, 24 
Quantitative tests for sugar in 
urine, 27 

Benedict's, 27 

fermentation, 28 
Quantity of urine, 22 

R 

Reaction of urine, 22 

Rectum, physical examination of, 21 

Red blood cells, in urinary sedi- 
ments, 36 

Red counting in blood, 40 

Robert's test for albumin in urine, 
23 

Rosenbach's test for bile in urine, 
31 

S 

Safranin, 83 

Sarcinse in gastric contents, 56 

Schmidt's test for bile pigments in 

feces, 60 
Sediment in urine, 22 



88 



Index 



►Serous exudates and transudates, 68 

cell count in, 69 

differential cell count in, 69 

examination of, 68 

microscopic examination of, 69 

staining for bacteria, 69 

staining for tubercle bacilli, 69 
Specific gravity of urine, 22 
Spinal fluid, 65 

cell count in, 65 

chemical tests, 68 

differential cell count, 66 

examination of, 65 

staining for bacteria, 67 
Sputum, 47 

Curschmann's spirals in, 47 

diphtheria bacilli in, 51 

diphtheria cultures, 51 

elastic fibers in, 47 

examination of, 47 

gross appearance of, 47 

influenza bacilli in, 50 

microscopic examination of, 47 

moulds in, 47 

parasites in, 47 

pneumococci in, 51 

stained, 47 

tubercle bacilli in, 48 

unstained, 47 
Staining for bacteria in serous exu- 
dates and transudates, 69 
Staining for bacteria in spinal fluid, 
67 

influenza bacilli, 67 

meningococci, 67 

pneumococci, 67 

staphylococci, 67 

streptococci, 67 

tubercle bacilli, 67 
Staining for tubercle bacilli in ser- 
ous exudates and transudates, 
69 
Stains used in the laboratory, 82 
Staphylococci in spinal fluid, 67 
Streptococci in spinal fluid, 67 
Sugar in urine, 25 

Benedict's test for. 26 

Fehling's test for, 25 

fermentation test for, 26 

quantitative tests for, 27 

T 

Test meals in examination of gas- 
tric contents, 56 
Transitional leucocytes, 45 
Trypsin in duodenal contents, 58 



Trypsin in feces, 62 
Tubercle bacilli: 

acid alcohol method for estimation 

of, in sputum, 48 
antiformin method for concentrat- 
ing, in sputum, 49 
in serous exudates and transu- 
dates 69 
in spinal fluid, 67 
in sputum, 48 
in urinary sediments, 36 
Pappenheim's method for estima- 
tion of, in sputum, 48 

TJ 

Urea, in urine, 33 

quantitative estimation of, by 
Doremus method, 33 
Urinary sediments, casts in, 36 

crystals in, 36 

gonococci in, 37 

leucocytes in, 36 

red blood cells in, 36 

tubercle bacilli in, 36 
Urine, 22 

acetone in, 29 

albumin in, 23 

Bence-Jones bodies in, 25 

beta-oxybutyric acid, 30 

bile in, 31 

blood in, 32 

color of, 22 

diacetic acid in, 30 

diazo reaction of, 30 

indican in, 31 

microscopic examination of sedi- 
ments in, 34 
. quantity of, 22 

reaction of, 22 

sediments in, 22 

specific gravity of, 22 

sugar in, 25 

urea in, 33 

W 

Wassermann reaction, 68, 70 

apparatus for, 71 

modifications of, 70 

reagents for, 71 . 

technic of, 75 
White counting, in blood, 38 
Widal reaction, 45 
Wright's stain, 83 



Yeasts in gastric contents, 56 



k 




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